Monocyte biomarkers for cancer detection

ABSTRACT

The disclosure relates to the field of biomarkers to diagnose a disease, more particularly to the field of biomarkers to diagnose cancer, and most particularly to colorectal cancer. Specifically, these biomarkers are expressed in monocytes of a subject, particularly circulating monocytes, as can be isolated from peripheral blood. The markers are particularly useful for early detection of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2013/051595, filed Jan. 28, 2013, designating the United States of America and published in English as International Patent Publication WO 2013/110817 A1 on Aug. 1, 2013, which claims the benefit under Article 8 of the Patent Cooperation Treaty to European Patent Application Serial No. 12152916.8, filed Jan. 27, 2012, the disclosure of each of which is hereby incorporated herein in its entirety by this reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)—SEQUENCE LISTING SUBMITTED AS PDF FILE WITH A REQUEST TO TRANSFER CRF FROM PARENT APPLICATION

Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing a PDF version of the Sequence Listing has been submitted concomitant with this application, the contents of which are hereby incorporated by reference. The transmittal documents of this application include a Request to Transfer CRF from the parent application.

TECHNICAL FIELD

The application relates to the field of biomarkers to diagnose a disease, more particularly to the field of biomarkers to diagnose cancer, most particularly colorectal cancer. Specifically, these biomarkers are expressed in monocytes of a subject, particularly circulating monocytes as can be isolated from whole blood. The markers are particularly useful for early detection of cancer.

BACKGROUND

Colorectal cancer (CRC) is the third most common cancer and the fourth cause of cancer-related deaths worldwide. Each year, almost one and a quarter million new cases of colorectal cancer are diagnosed and over six hundred thousand deaths are attributable to this disease (Ferlay et al., 2010). Its incidence and difficult early diagnosis make CRC a primary focus in the oncology community. CRC is often diagnosed after symptoms appear, but most people with early disease are symptomless and symptoms usually appear only with more advanced disease. Therefore, 30 to 40% of CRC patients have metastatic disease at diagnosis (Benson, 2007). The overall survival rate of patients with metastatic colorectal cancer at 5 years is less than 10%. In contrast, up to 80% of patients can be cured by tumor resection if the CRC is detected at an early stage (Benson, 2007). Therefore, early diagnosis is important for the proper control of CRC. Although colonoscopic screening for CRC is currently the most reliable screening tool, the invasive nature and the incurred cost have hampered the wide application of this procedure. On the other hand, the fecal occult blood testing is a non-invasive alternative. However, it lacks the sensitivity and specificity required for an effective screening tool and requires meticulous dietary restriction.

Unfortunately, current therapeutic approaches also lack efficacy to successfully cure the disease, especially in patients with metastatic CRC. Therefore, much effort is needed to optimize the treatment of CRC. In 2004, the U.S. Food and Drug Administration (FDA) approved bevacizumab (AVASTIN®), a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), in combination with classic chemotherapy as a first line treatment for CRC. However, the phase 3 clinical trial revealed that this combination therapy only adds less than 5 months to the overall survival of CRC patients (Hurwitz et al., 2004).

Thus, there is a pressing need to identify specific non-invasive biomarkers for CRC early diagnosis and monitoring, in order to avoid the more advanced stages of the disease that are difficult to cure. Such early, non-invasive biomarkers would in fact be useful for several other tumor types as well.

DISCLOSURE

Provided are biomarkers that are both non-invasive and specific to the disease. Envisaged diseases are different types of cancers, such as (but not limited to) gastric cancer and colorectal cancer, and inflammatory bowel disease (IBD). Most particularly, the biomarkers are biomarkers for colorectal cancer (CRC).

Inflammatory cells such as macrophages and dendritic cells have been suggested as potential (source of) biomarkers in different cancers, as they infiltrate in the tumor tissue. However, they have several drawbacks: due to the dual role of distinct subpopulations of macrophages on tumor growth and progression according to their functional polarization, macrophages have been implicated both in tumor growth and improved survival. Likewise, different status of dendritic cell maturation can correlate with both bad and good prognosis of CRC. Thus, their suitability as marker is unclear. Moreover, since they are in tissue, isolating the markers is an invasive procedure. As they also require the presence of a tumor large enough to isolate them from, they are not suited as early markers.

Thus, it was explored whether precursors of these inflammatory cells could serve as markers. Peripheral blood is one of the less invasive sample sources that can be intensively screened for CRC biomarkers. Blood can for instance be screened for carcinoembryonic antigen (CEA), but this test has a suboptimal sensitivity and specificity. Alternatively, circulating tumor cells can be measured, but this has the drawback of having a low detection rate. It is also not suited for very early detection of cancer. Other blood tests are currently being developed, but often have issues with sensitivity and/or specificity. This is not surprising, given the heterogeneous composition of blood and the opposing functions of its cellular components during cancer progression.

The cellular components of peripheral blood include red blood cells, white blood cells, and platelets. White blood cells, or leukocytes, are cells of the immune system involved in defending the body against both infectious disease and foreign materials. Five different and diverse types of leukocytes exist: neutrophils, basophils, and eosinophils (together the granulocytes or polymorphonuclear leukocytes), lymphocytes and monocytes (together the agranulocytes, mononuclear leukocytes or peripheral blood mononuclear cells (PBMCs)). The three major types of lymphocyte (all from the lymphoid lineage) are T cells and B cells (the major cellular components of the adaptive immune response) and natural killer (NK) cells. Monocytes (from the myeloid lineage) are part of the innate immune system and are able to migrate quickly to a site of inflammation in the body.

Circulating monocytes present in whole blood can give rise to macrophages and dendritic cells. It was investigated whether these monocytes are modified when they are circulating in the blood stream, and whether these cells can be used as diagnostic tools. Surprisingly, it was found that in cancer patients, from a very early stage of the disease, there is a small, yet distinct and reproducible shift in expressed gene signature of circulating monocytes. This is especially surprising, given that circulating monocytes, contrary to tumor-associated macrophages or infiltrated dendritic cells, are not in direct contact with the tumor. Indeed, in circulation they are at a considerable distance from the actual tumor site and they are separated from the tumor by endothelial cells composing tumor blood vessels. This “distant education” thus is different from expression profile changes upon close contact with the tumor, as seen, e.g., in resident macrophages. Without being bound to a particular mechanism, it seems plausible that the observed differences in gene expression of circulating monocytes are caused by factors released by the tumor, such as (but not limited to) cytokines. These differences in monocyte gene expression can be used as biomarkers for cancer, which are both very specific and very sensitive. Importantly, the biomarker(s) can be obtained in a minimally invasive way, as only a small sample of (typically peripheral) blood is required.

Moreover, a subset of genes in this gene signature can be used to discriminate between patients with non-metastatic and metastatic disease. This allows stratification of patients, and may help guide decisions on suitable therapies.

Thus, according to a first aspect, methods are provided of diagnosing a disease in a subject, comprising determining the expression of at least one gene product in a sample of monocytes of said subject. Typically, the methods will be performed in vitro.

It is particularly envisaged that the disease is cancer, particularly a cancer that presents as a solid tumor, more particularly colorectal cancer. The concept of an altered gene signature in monocytes likely is applicable to numerous diseases with an inflammatory component, such as different types of cancer, or such as IBD (inflammatory bowel disease). However, different diseases likely will affect gene expression differently. For instance, it should be realized that the nature of the tumor is bound to influence the factors governing monocyte gene expression, as is shown in the Examples section for colorectal and gastric cancer. The underlying concept, of circulating monocytes whose gene expression profile is altered due to presence of a tumor or disease, is the same however. The signature presented herein is particular for colorectal cancer.

According to further embodiments, the at least one gene product whose expression is deter pined is not a gene product that is used as marker to identify the nature of monocytes in said sample. This in order not to create a bias: if, e.g., CD14 is first used as a marker to isolate monocytes, one has already enriched the population of CD14-expressing cells. This also applies for markers used for subset identification (e.g., Tie2 to isolate Tie2-expressing monocytes). Stated generally, in particular embodiments, any marker that has been used in the isolation procedure prior to determining gene expression of the at least one gene in the sample of monocytes is not envisaged as marker whose expression is determined.

According to further embodiments, the monocytes are circulating monocytes.

According to further embodiments, altered expression of the at least one gene product is indicative of the disease, particularly of cancer; i.e., altered expression of the at least one gene product in a sample of monocytes of a subject is indicative of the presence of a cancer in the subject. Altered expression can be increased or decreased expression. This will depend on the selected genes, as some will be up-regulated while others are down-regulated. Moreover, some genes will show altered expression depending on disease stage, e.g., altered expression between the non-metastatic and metastatic stage of cancer.

If the disease is cancer, it is particularly envisaged that it is colorectal cancer.

For embodiments relating to cancer detection, the monocyte expression profiling described herein is particularly well suited for early detection of cancer. For example, for colon cancer, early detection means that the monocyte expression is already altered (and thus the tumor may be detected) when the tumor is still in Tis stage (i.e., in situ, the tumor only affects the mucosa) or in T1 stage (submucosal infiltration, but not further). This is a particular advantage, since these tumors are routinely missed using other procedures. Without being bound to a particular theory, it is shown herein that the signature (i.e., the altered gene expression is specific to the tumor and not hypoxia-dependent. This may be the reason why smaller, non-hypoxic tumors can be detected using the methods described herein.

According to further embodiments, the at least one gene product comprises at least one product selected from the genes listed in Table I. According to yet further specific embodiments, the at least one gene product comprises at least one product selected from the genes listed in Table II. According to alternative specific embodiments, the at least one gene product comprises at least one product selected from the genes listed in Table IV.

According to specific embodiments, the at least one gene product comprises at least one product selected from the following genes: ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF. Alternatively, or additionally, the selection is made from the list of: ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, LOC644063, S100P, SEPT5, SLPI, SOCS3, and TKT. More particularly, the at least one gene product comprises at least one product selected from the following genes: ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, S100P, SOCS3, TKT, and TNF. A further particular list from which at least one gene product may be selected is ACP5, ADM, APP, CD68, CTSZ, DDIT4, FKBP5, GPER, HBB, HP, LAPTM4A, S100P, SOCS3, and TKT; or alternatively: ACP5, ADM, APP, CD68, CTSZ, FKBP5, GPER, HBB, HP, LAPTM4A, S100P, SOCS3, and TKT. Yet another particular list from which at least one gene product may be selected is ADM, BAX, CD68, CTSZ, CXCR4, FKBP5, GPER, HBA1, HBB, LAPTM4A, S100P and TKT.

According to further embodiments, the at least one gene product is at least two gene products, more particularly at least three, four or five gene products, or even more particularly, at least six, seven, eight, ten, twelve, sixteen or twenty gene products. According to further embodiments according to this aspect, at least one gene product is from a gene selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF; and at least one other gene product is selected from the genes listed in Table I, Table II, Table III and/or Table IV. Alternatively, at least one gene product is from a gene selected from the list of: ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, LOC644063, S100P, SEPT5, SLPI, SOCS3, and TKT; and at least one other gene product is selected from the genes listed in Table I, Table II, Table III and/or Table IV.

In particular embodiments, primary cancer can be discriminated from metastatic cancer, using specific marker genes. According to specific embodiments, at least one of the gene products is chosen from ACP5, ADM, ALDH1A1, APP, CD68, ENSA, FKBP5, GPER, HLA-DQA1, LOC644063, LOC723972, RN28S1, S100P, TAF15, TKT and TNF (or alternatively, from ADM, BAX, CD68, CTSZ, CXCR4, FKBP5, GPER, HBA1, HBB, LAPTM4A, S100P and TKT). Note that in these instances, it is particularly envisaged to use more than one marker. For example, at least one marker may be chosen to discriminate between healthy subjects or subjects with disease, while at least one marker is chosen to discriminate between patients with primary cancer or metastasized cancer.

According to specific embodiments, the monocytes have been isolated from a blood sample (e.g., peripheral or central blood sample). It is particularly envisaged that the blood sample is obtained no more than 12 hours, no more than 10 hours, no more than 8 hours, no more than 6 hours, no more than 4 hours or no more than 2 hours prior to starting the isolation of the monocytes from the blood sample.

In a further aspect, methods are provided for diagnosing cancer in a subject, comprising the steps of:

-   -   Isolating, from a blood sample of said subject, a monocyte         population;     -   Determining in said monocyte population the expression or levels         of expression of at least one gene product;     -   Correlating the expression of the at least one gene product with         the presence or absence of cancer in said subject.

According to further embodiments, the isolating step is done within 12 hours of obtaining the blood sample from the subject. Particularly, however, the isolation of the monocytes is done as soon as possible after drawing the blood from the subject, e.g., within 10 hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours, within 1 hour, or immediately following the blood collection. The isolation of the monocytes should ideally be finished within 12 hours, more particularly within 10 hours, 9 hours, 8 hours, 7 hours, or most particularly within 6 hours of obtaining the blood sample from the subject.

Determining expression or levels of expression may be done using any suitable technique known to the skilled person. When done at the RNA level, suitable techniques include, but are not limited to, RT-PCR (particularly RT-qPCR), hybridization microarray, SAGE (serial analysis of gene expression), RNA-Seq, or the nanostring technology (based on molecular barcoding). According to particular embodiments, the determining of expression levels is done with RT-PCR.

Suitable techniques to determine expression or levels of expression at the protein level include, but are not limited to, antibody-based detection methods such as Western blot, ELISA or FACS. Many of these techniques can also be done using alternatives to classical antibodies, e.g., nanobodies (single domain antibodies, developed by Ablynx), alphabodies (single-chain, triple-stranded coiled coil proteins, developed by Complix) or other protein-binding molecules.

The methods described herein are also particularly useful to monitor evolution of the disease. This is because the gene signature retains plasticity. Monocytes will show an altered gene expression pattern quite rapidly (within hours rather than days) when exposed to an inflammatory stimulus in the body (e.g., the presence of cancer, such as colorectal cancer. This will happen even if the tumor is very small). However, when the stimulus is taken away (e.g., by surgically resecting the tumor), the monocytes will revert their gene expression to baseline values, and vice versa when the stimulus is introduced again.

Thus, according to particular embodiments, the methods provided herein are used for monitoring recurrence of a cancer. In other words, the subject from whom the monocytes are obtained is a subject that had cancer, but in which the cancer has been removed. The methods are then performed to evaluate whether the treatment of the cancer has been successful, or whether the tumor has returned. This is particularly envisaged for monitoring subjects who are at high risk of recurrence (for example, patients who had poorly differentiated tumors or venous or lymphatic invasion).

According to alternative, but not exclusive, particular embodiments, the methods are used for monitoring response to therapy. For example, once the subject has been diagnosed with cancer (using the methods described herein or alternative methods known in the art), and treatment for cancer has initiated, the present methods are performed again over time, to see whether the monocyte gene expression returns to baseline values (i.e., levels comparable to those found in healthy volunteers), as this would be indicative of a successful treatment and eradication of the tumor. It is envisaged that the present methods are particularly suited in determining minimal residual disease (MRD). MRD is the name given to small numbers of cancer cells that remain in the patient during treatment, or after treatment when the patient is in remission (no symptoms or signs of disease). In cancer treatment, MRD testing has several important roles: determining whether treatment has eradicated the cancer or whether traces remain, comparing the efficacy of different treatments, monitoring patient remission status and recurrence of the cancer and choosing the treatment that will best meet those needs (personalization of treatment). Given the fact that even very small tumors are detected with the present gene signature, it is particularly envisaged that the signature presented herein is highly suitable for MRD testing even when the patient has no other symptoms or signs of disease.

As the altered gene expression correlates to the presence of disease and can be used to monitor response to therapy, it is also envisaged that the methods provided herein may comprise an additional step of changing the treatment of the patient based on the results of the diagnosis. For instance, if the patient is receiving a combination of chemotherapeutics, but this does not result in gene expression in the monocytes reverting back to baseline values, this is indicative that the therapy does not work, and a decision may be taken to switch to a different chemotherapy regimen (e.g., different or additional drugs, or a different dose of the same drugs).

According to a further aspect of the disclosure, a gene signature is provided. This gene signature typically comprises at least two genes, wherein at least one gene is selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF; and at least one other gene is selected from the genes listed in Tables I, II, III and/or IV.

According to most specific embodiments, the gene signature is a monocyte gene signature, i.e., a group of genes with altered expression in monocytes, characteristic for a particular condition (particular cancer, most particular colorectal cancer).

The gene signature is particularly useful in the field of medicine. Thus, according to specific embodiments, the gene signature provided herein is provided for use as a medicament. Particularly, it is provided for use as a diagnostic. More particularly, the gene signature provided herein is provided for use in the diagnosis of cancer. Most particularly, the gene signature provided herein is provided for use in the diagnosis of colorectal cancer.

According to a further aspect, kits are provided comprising means to detect the gene signature. For example, kits are provided that contain means to detect expression or levels of expression of at least two genes, wherein at least one gene is selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF; and at least one other gene is selected from the genes listed in Tables I, II, III and/or IV.

It is envisaged that these kits are provided for a particular purpose, such as for diagnosis of cancer, or more particularly, for the diagnosis of colorectal cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1. ROC curve for several classification approaches to discriminate patient samples (both metastatic and non-metastatic patients, P, PM) from samples obtained from healthy volunteers (HV). A: Limma wrapper approaches, B: DEDS wrapper approaches, C: All embedded approaches, D: the gene signature from the original Limma analysis as comparison.

FIG. 2. mRNA expression levels for selected genes in a random subset of samples (n=8). Left panels show mean expression values and standard error of the mean, middle panels show Box-and-Whiskers Plot of the same data sets (dark line in middle is median value, top and bottom edges of the rectangles respectively represent the 75th and 25th percentile values, top and bottom lines are maximum and minimum values), right panels show the ROC curve (plot of the sensitivity, or true positive rate, vs. false positive rate (1—specificity or 1—true negative rate)). Normalization occurred against beta-2-microglobulin (B2M) levels (shown for most molecules) or actin expression levels (shown for DNAJC7, FCER1A, HLA-DQA1, LOC723972, and RN28S1), expression values are shown as copies/10 000 copies of B2M or actin. HV, healthy volunteers. P, patients with a primary tumor without evidence of metastatic disease. PM, patients with a metastasized tumor. P, PM: P and PM groups combined. p values in the left panel are calculated by 2-way ANOVA for differences between HV, P, and PM (top left corner) and by Student's two-sided t-test with Welch's correction for differences between HV and P, PM (below the bars). p values in the middle panel are calculated by Student's two-sided t-test with Welch's correction for differences between P and PM. p values in right panel are indicated for AUC.

FIG. 3. Relative expression of DDIT4 in mice with (early and advanced cancer) and without (precursor, colitis) chemically induced neoplasms.

FIG. 4. mRNA expression levels for selected genes from a multicenter clinical study. A, Left panels show mean expression values and standard error of the mean; right panels show the ROC curve (plot of the sensitivity, or true positive rate, vs. false positive rate (1—specificity or 1—true negative rate)). Normalization occurred against beta-2-microglobulin (B2M) levels, expression values are shown as copies/10 000 copies of B2M. HV, healthy volunteers. P, PM: combination of patients with a primary tumor (P) and patients with a metastasized tumor (PM). p values in the left panel are calculated by Student's two-sided t-test with Welch's correction for differences between HV and P, PM. p values in right panel are indicated for AUC. B, ROC curve for selected genes from a multicenter clinical study, comparing the combined P, PM groups to the HV group. Normalization occurred against beta-2-microglobulin (B2M) levels, expression values are shown as copies/10 000 copies of B2M. Small differences between the ROC curves of FIGS. 4A and 4B are due to the random split in training and test set.

FIG. 5. ROC values for selected gene signatures from a multicenter clinical study for the combined P and PM groups vs. the HV group. Left panel shows the ROC for the combined 43 genes of Table IV, using normalization against B2M levels as described for FIG. 4. Red line, Single Gene Majority Vote (SGVM) classification: AUC=0.912; BER=0.204. Blue line, Support Vector Machine (SVM) classification: AUC=0.917; BER=0.151. Right panel shows the ROC for a subset of 12 genes of Table IV (ADM, BAX, CD68, CTSZ, CXCR4, FKBP5, GPER, HBA1, HBB, LAPTM4A, S100P, and TKT). Relative normalization was used (i.e., each individual expression value is normalized to the average of the HV group). Red line, SGVM classification: AUC=0.915; BER=0.156; Blue line, SVM classification: AUC=0.903, BER=0.18.

FIG. 6. mRNA expression levels for selected genes from a multicenter clinical study. Plots show mean expression values and standard error for the depicted genes. Left boxes are the HV group, right boxes are the combined P, PM groups. Normalization occurred against beta-2-microglobulin (B2M) levels, expression values are shown as copies/10 000 copies of B2M.

FIG. 7. Comparison of expression of selected genes in monocytes obtained from colorectal cancer patients (CRC, dark grey bars on the left) or gastric cancer patients (GC, light grey bars on the right). Expression shown as fold change relative to monocytes obtained from healthy volunteers. The genes selected all have a p value <0.01 for colorectal cancer patients. Genes significantly regulated in gastric cancer patient monocytes compared to healthy volunteers are denoted with an asterisk (p<0.05).

FIG. 8. Comparison of expression of selected genes in monocytes obtained from healthy volunteers cultured with normal medium (mock), medium conditioned by colorectal tumor cell line HCT116 (HCT Norm) or medium conditioned by the gastric tumor cell line MKN (MKN).

FIG. 9. Comparison of expression of selected genes in monocytes obtained from healthy volunteers cultured with normal medium, medium conditioned by a primary colonic epithelial cell line (CCD), medium conditioned by colorectal tumor cell line HCT116 in normoxic (HCT Norm) or hypoxic (HCT hyp) conditions.

FIG. 10. Reversal of the gene signature. Monocytes from healthy volunteers were incubated with medium conditioned by a primary colonic epithelial cell line (CCD) (1^(st) box and whisker, in white) or medium conditioned by colorectal tumor cell line HCT116 in normoxic conditions for 18 hours (2^(nd) box and whisker, dark grey), after which the medium was refreshed to plain culture medium, and the gene signature was assessed again after 72 hours (3^(rd) and 4^(th) box and whisker respectively, in white for the non-tumor exposed cells; in grey for the monocytes earlier exposed to HCT-conditioned medium).

DETAILED DESCRIPTION Definitions

The disclosure will be described with respect to particular embodiments and with reference to certain drawings but the disclosure is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term “comprising” is used in the description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun, e.g., “a” or “an,” “the,” this includes a plural of that noun unless something else is specifically stated.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the disclosure described herein are capable of operation in other sequences than described or illustrated herein.

The following terms or definitions are provided solely to aid in the understanding of the disclosure. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the disclosure. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 3^(rd) ed., Cold Spring Harbor Press, Plainsview, N.Y. (2001); and Ausubel et al., Current Protocols in Molecular Biology (updated till Supplement 96), John Wiley & Sons, New York (2011), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

As used herein, the noun “subject” refers to an individual vertebrate, particularly a jawed vertebrate (i.e., member of the Gnathostomata, which are characterized by a particular adaptive immune system), more particularly an individual mammal, most particularly an individual human being. A “subject” as used herein is typically a human, but can also be a mammal, particularly domestic animals such as cats, dogs, rabbits, guinea pigs, ferrets, rats, mice, and the like, or farm animals like horses, cows, pigs, goat, sheep, llamas, and the like. A subject can also be a non-mammalian vertebrate, like a fish, reptile, amphibian or bird; in essence any animal that has a circulatory blood system wherein monocytes are part of the content of the blood fulfills the definition of subject herein.

“Expression” or “expression of a gene product” as used in the application refers to the process by which inheritable information from a gene, such as the DNA sequence, is made into a functional gene product, such as protein or RNA. This definition thus encompasses, but is not limited to, transcription and/or translation of a gene. “Determining expression” may encompass processes such as detecting or measuring the presence of gene products, or determining the expression levels, i.e., the (relative or absolute) amount of gene product present. Determining expression may be done qualitatively (i.e., whether or not there is expression in a sample) and/or quantitatively (determining the amount of expression, or expression levels). Most typically, expression will be done quantitatively, in order to be able to compare expression levels. Determining expression may involve comparison with a positive control (e.g., to assess whether gene products can be detected in the sample, in particular whether the detection method works), a negative control or a blank (typically to assess whether no false positive signal is being generated), one or more standards (either internal or external standards, typically to allow more accurate quantification), or a combination thereof. The positive control may additionally or alternatively be an internal positive control, typically a gene product known to be present in the sample (e.g., to assess whether gene products can be detected in the sample, in particular whether the detection method works or whether gene products are indeed present in the sample). Detection of expression and/or activity is well known in the art, and a skilled person is capable of choosing appropriate controls and/or standards.

A “gene product” as used herein typically refers to what is transcribed or translated from the gene in question, such as mRNA and protein. The different isoforms or variants of mRNA and the resulting protein isoforms or variants are envisaged within the term gene product. Fragments of a gene product are also envisaged, as long as they are functionally active.

When mRNA is chosen as the (or one of the) gene product whose levels are determined, this can be the total of all mRNA isoforms for the gene(s) under study, or one or more specific mRNAs, e.g., only those expressed in monocytes.

Alternatively or additionally, the gene product of which the levels are determined may be protein. As protein is translated from mRNA and the mRNA exists in multiple isoforms, the same considerations apply: the total protein levels may be determined, or those of specific isoforms only (e.g., using an antibody against the different C-termini). Most particularly, all protein isoforms may be detected (e.g., using an antibody against a common epitope). Of note, it is envisaged as well that both mRNA and protein are determined. In this case, the isoforms to be detected can be all isoforms for both mRNA and protein, identical isoforms (wholly overlapping), or different isoforms (partly or not overlapping), depending on the setup of the experiment. With identical isoforms, it is meant that the mRNA isoform encodes for the corresponding protein isoform. However, for several genes it is known that the number of protein isoforms detected is generally lower than the number of possible mRNA transcripts (and thus of protein isoforms).

Also envisaged is the detection of protein with specific post-translational modifications, either within the whole protein pool or through the selective detection of such modified proteins. Examples of such modified proteins include, but are not limited to, methylated, phosphorylated, ubiquitinylated, glycosylated proteins or any combination thereof.

The term “monocytes” as used herein refers to a particular type of white blood cells that are part of the innate immune system of vertebrates. Monocytes are normally produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. They circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. Monocytes constitute between three to eight percent of the leukocytes in the blood (reference values in healthy adult humans), and are the largest corpuscle in blood. Once extravasated from the bloodstream to other tissues, they will differentiate into tissue resident macrophages or dendritic cells. The different types of monocytes in human blood can be characterized by expression of CD14 and CD16 cell surface markers: there are CD14^(hi) CD16^(−/lo) monocytes (also termed “classical” monocytes), CD 14^(lo) CD 16^(hi/+) monocytes (“non-classical monocytes”). Sometimes a third, intermediate category is also distinguished (CD14^(hi) CD16^(lo)).

In humans, CD14 is considered a marker of the monocyte lineage. So, at least in humans, “monocytes” can be considered equivalent to CD 14-expressing cells that circulate in the bloodstream (the latter property distinguishing them from dendritic cells and macrophages). Although virtually all CD14-expressing cells in peripheral blood will be monocytes, further differentiation using other markers or cell size can be made to distinguish monocytes from other cell types.

The terms “circulating blood” or “whole blood,” which are used as equivalents herein, refer to the blood within the closed cardiovascular system of vertebrates. This comprises the blood in arteries (arterial blood), in veins (venous blood) as well as capillaries, venules and portal systems. Circulating blood cells are the cellular components of blood, consisting of red blood cells, white blood cells, and platelets, which are found within the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver, or bone marrow. The term “whole blood” encompasses both peripheral blood, found in the systemic circulation, and central blood, present in the pulmonary and coronary circulation. The term “circulating monocytes” refers to monocytes that are present in the (peripheral or central) blood and thus part of the circulation (and not migrated into tissue). A “sample of monocytes” as used herein refers to a sample wherein the majority, particularly the large majority, of cells are monocytes. Preferably, the sample consists essentially of monocytes, or it consists exclusively of monocytes. Since only monocytes give rise to macrophages or dendritic cells (the myeloid lineage), and it is desirable that the sample represents this lineage, it is particularly envisaged that the sample contains as little other blood cells as feasible (preferably none). Thus, it is envisaged that no (or virtually none, or as little as feasible) other leukocytes are present in the sample, that no (or virtually none, or as little as feasible) granulocytes are present in the sample, that no (or virtually none, or as little as feasible) other PBMCs are present in the sample, that no (or virtually none, or as little as feasible) lymphocytes (T cells, B cells, NK cells) are present in the sample. According to particular embodiments, the sample does not consist of a mixed monocyte-lymphocyte population. In other words, the sample is not a population of peripheral blood mononuclear cells. According to alternative embodiments, the sample does not contain lymphocytes. Thus, a sample of monocytes can be interpreted as a sample of isolated monocytes. In other words, although circulating blood contains monocytes, according to particular embodiments, it does not fulfill the definition of a sample of monocytes. To obtain a sample of monocytes from a blood sample, a further separation or isolation step is needed. Typically, this is done by density gradient separation (to isolate the PBMNC from the rest of the blood constituents) followed by marker-assisted separation; but a one-step isolation procedure (by marker(s) only) can be applied as well. For example, in humans, one can separate the CD14-expressing cells in peripheral blood from the non-CD 14-expressing cells to obtain a sample of monocytes as envisaged herein. Although monocytes can be further divided in subpopulations, for instance depending on expression of particular receptors (e.g., but not limited to, CD16), it is primarily envisaged herein to use a sample of all subtypes of monocytes found in the peripheral blood. According to alternative embodiments, gene expression can be determined in particular subsets of monocytes (e.g., CD16-expressing monocytes, Tie-2 expressing monocytes, CCR2-expressing monocytes, etc.). However, according to very specific embodiments, the methods presented herein are not practiced on a sample consisting essentially of a subset of Tie2-expressing monocytes.

Further, since the present gene signature is particularly envisaged for making a diagnosis, it is particularly foreseen that freshly isolated monocytes are used. With “freshly isolated” it is meant that the monocytes have not been kept in culture, since this would influence gene expression as well as other properties (Hart et al., 1995; see Example section as well). Importantly, it does not necessarily imply that the monocytes have been isolated immediately prior to determining the gene expression, as it is possible (and often more practical) to isolate the monocytes beforehand, store them (e.g., by lysing them and freezing them) and determine gene expression at a later point in time. Thus, according to particular embodiments, the sample of monocytes does not consist of cultured monocytes (or a culture of monocyte cells).

As is evident from the previous paragraphs, the sample of monocytes may be provided as such, or may be pre-processed. Examples of pre-processing include, e.g., lysis or additional separation or purification steps.

As used herein, “altered levels” of a gene product may mean either “increased levels” or “decreased levels” of a gene product, which is typically assessed versus a control. The skilled person is capable of picking the most relevant control. This may for instance depend on the particular gene product, the nature of the disease or cancer studied, the sample(s) that is/are available, and so on. Suitable controls include, but are not limited to, expression in a sample of monocytes of a subject that is cancer-free (optionally from the same subject when he/she was still healthy), a monocyte sample from a subject with a non-metastasizing tumor (particularly with the same tumor type as the subject, e.g., colorectal cancer), or a set of clinical data on average monocyte gene product levels in healthy volunteers or patients with non-metastasizing tumors. It may also be an artificially generated expression standard, e.g., as used in “real” quantitative PCR. As is evident from the foregoing, the control may be from the same subject, or from one or more different subjects or derived from clinical data. Optionally, the control is matched for, e.g., sex, age, etc.

With “increased” levels of a gene product as mentioned herein, it is meant levels that are higher than are normally present. Typically, this can be assessed by comparing to control. According to particular embodiments, increased levels of a gene product are levels that are 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 100%, 150%, 200% or even more higher than those of the control. According to further particular embodiments, it means that the gene product is present, whereas it normally (or in control) is not expressed, or is expressed at very low or barely detectable levels. In other words, in these embodiments detecting the presence of a particular gene product is equivalent to detecting increased levels of the gene product. According to yet further particular embodiments, it means that the gene product is present, whereas in the majority of monocyte samples from tumor-free individuals, taken as a control, it is not. The skilled person will appreciate that the exact levels by which a gene product needs to be higher in order to allow a reliable and reproducible diagnosis may depend on the type of tumor tested, of which product (mRNA, protein) the levels are assessed and the natural variability of these levels. However, assessing the increase itself is fairly straightforward, since it only requires routine techniques.

Instead of looking at increased levels compared to a negative control, the skilled person will appreciate that the reverse, comparing to a positive control, can also be done. Thus, if the gene product levels measured in the monocyte sample are similar to those of a “control” obtained from a subject with a tumor, or with a metastasizing tumor (or are, e.g., comparable to monocyte gene product levels found in a clinical data set of tumors or metastasizing tumors), this may be considered equivalent to increased gene product levels compared to a negative control, and be correlated to presence of disease, or progression to metastasis. In the other case, if gene product levels are significantly lower than those of a positive control, this can be used to establish the absence of a metastasizing tumor, or even the absence of a tumor as such.

For “decreased levels” of a gene product compared to a positive control, the considerations about increased levels of a gene product apply mutatis mutandis. Of course, gene product levels may be compared to both a negative and a positive control in order to increase accuracy of the diagnosis.

Note that, when both increase in levels of a gene product and decrease in levels of another gene product are determined, the levels of one gene product can be divided by the levels of the other gene product. When comparing the resulting value to that of a division of the appropriate control or standard values, the potential difference will be enlarged when both genes have an altered expression, thereby enlarging the window for detection.

The term “cancer” as used herein, refers to different diseases involving unregulated cell growth, also referred to as malignant neoplasm. The term “tumor” is used as a synonym in the application. It is envisaged that this term covers all solid tumor types (carcinoma, sarcoma, and blastoma). According to particular embodiments, the cancer is not a liquid tumor (blood cancer). According to specific embodiments, the cancer is not leukemia, not lymphoma and/or not myeloma. The term “colorectal cancer” as used herein is meant to include malignant neoplasms of colon (C18 in ICD-10), malignant neoplasms of rectosigmoid junction (C19 in ICD-10), malignant neoplasms of rectum (C20 in ICD-10) and malignant neoplasms of anus and anal canal (C21 in ICD-10).

“Early detection” of disease refers to establishing the presence of a disease before it has fully developed. Many diseases are classified in grades, stages or classes to describe the different phases of a disease. For example, for cancer, cancer stages can be classified using the TNM system or Roman numeral staging according to UICC or AJCC, respectively. In the TNM systems, detection of tumors that are in situ (“Tis”) or are sufficiently small (typically in the magnitude of 1 cm, although this depends on tumor type and affected organ) and extend minimally to other tissues (“T1”) whilst they have not spread to lymph nodes (“N0”) and are not metastasized (“M0”) can be considered as early detection. In the Roman numeral staging, this corresponds to detection of Stage 0 or Stage I tumors. Other classifications exist, often depending on tumor type (e.g., Dukes staging for colon cancer, CIN grading for cervical cancer, etc.). The skilled person will be aware of the features for each tumor type that are typical for early stages, and of the stages in other systems that correspond to Tis, T1, Stage 0 or Stage I tumors.

A “gene signature” as used herein refers to a group of genes in a type of cell whose combined expression pattern is uniquely characteristic of a medical or other condition. Typically, a group of genes refers to at least two genes, but this may also be at least three genes, at least four genes, at least five genes, at least six genes, at least seven genes, or at least eight genes. Sometimes, it may even be at least more than eight genes (e.g., 10 genes, 12 genes, 16 genes, 20 genes, etc.). According to particular embodiments, the gene signature is a monocyte gene signature, i.e., a gene signature in monocytes. According to particular embodiments, the gene signature is characteristic of cancer, particularly of colorectal cancer.

Peripheral blood is one of the less invasive sample sources that can be intensively screened for CRC biomarkers. Peripheral blood monocytes (PBM) represent a reservoir of inflammatory cells that contribute to disease progression by different means (Murdoch et al., 2008). When recruited into tumors from the circulation, the microenvironment can drive monocyte differentiation into either macrophages or dendritic cells. Tumor-associated macrophages (TAMs) are typically characterized as either “classically” activated tumoricidal and anti-angiogenic macrophages (termed M1) or “alternatively” activated pro-tumorigenic and pro-angiogenic macrophages (termed M2). Monocyte-derived dendritic cells (DCs) are professional antigen-presenting cells that raise the immune response against the tumor. However, the tumor restricts the development of fully competent mature DCs. As a consequence, immature DCs display low immune-stimulatory capacity and induce immune-tolerance. In CRC, the contribution of TAMs to the progression of the disease is controversial as high numbers of infiltrating tumor associated macrophages have been associated with both tumor growth (Sickert et al., 2005) and improved disease-free survival (Forssell et al., 2007). The different outcomes may be explained by the dual role of distinct subpopulations of macrophages on tumor growth and progression according to their functional polarization (Lewis and Pollard, 2006; Allavena et al., 2008). Similarly, different status of dendritic cell maturation can correlate with both bad and good prognosis of CRC patients (Sandel et al., 2005). However, it remains unknown in the art whether PBM, cell precursors of TAMs and DCs, are modified by tumor released cytokines when they are circulating in the blood stream, and whether these cells can be thus used as diagnostic tools.

As detailed herein, it is surprisingly shown that circulating monocytes indeed show an altered gene expression pattern from the very earliest stages of the presence of disease, such as presence of a tumor. It is particularly envisaged that the disease is an inflammatory disease or disease with an inflammatory component, given the roles of monocytes in the inflammation process. Examples of inflammatory diseases include, but are not limited to: atherosclerosis, acne vulgaris, allergies, asthma, autoimmune diseases, cancer, celiac disease, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, ischemic heart disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, and interstitial cystitis. In principle, for each of these inflammatory diseases a monocyte gene signature can be derived, comprising at least one gene differentially regulated in monocytes.

A most particular envisaged inflammatory disease is cancer. A most particularly envisaged form of cancer is colorectal cancer. Thus, methods of diagnosing diseases such as cancer in a subject are provided, comprising determining the expression of at least one gene product in a sample of monocytes of said subject.

The skilled person is well aware of different methodologies that can be used to determine expression of at least one gene product, in particular, for quantitative measuring of the gene product.

When the gene product is mRNA, for instance, levels of mRNA can be quantitatively measured by Northern blotting, which gives size and sequence information about the mRNA molecules.

A more modem approach for measuring mRNA abundance is reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR). RT-PCR first generates a DNA template from the mRNA by reverse transcription, which is called cDNA. This cDNA template is then used for qPCR where the change in fluorescence of a probe changes as the DNA amplification process progresses. With a carefully constructed standard curve qPCR can produce an absolute measurement such as number of copies of mRNA, typically in units of copies per nanoliter of homogenized tissue or copies per cell, or normalized as copies per specified number of copies for a housekeeping gene. Examples of housekeeping genes include, but are not limited to, actin and B2M. Although there may be slight (particularly quantitative) shifts when one uses normalization with one particular housekeeping gene over the other, the overall conclusion of (qualitative) up-regulation or down-regulation will not be affected. Any suitable housekeeping gene known to the skilled person is envisaged herein for use in normalization. It is also envisaged that normalization is done with more than one housekeeping gene, e.g., to check or improve robustness of results. qPCR has the advantage of being very sensitive (detection of a single mRNA molecule is possible).

If, rather than determining expression of one or a few genes, expression levels of multiple genes are simultaneously determined, DNA microarrays are a suitable technique. Using DNA microarrays, transcript levels for many genes at once (expression profiling) can be measured. Recent advances in microarray technology even allow for the quantification, on a single array, of transcript levels for every known gene in several organism's genomes, including humans.

An alternative way of measuring mRNA abundance makes use of “tag based” technologies. An example includes SAGE (serial analysis of gene expression), which extracts small chunks of sequences from mRNA molecules, links these into concatemers and sequences these.

Another alternative is RNAseq, also known as whole transcriptome shotgun sequencing, which uses high-throughput next-generation sequencing technologies to sequence cDNA in order to analyze a sample's RNA content. Examples of next-generation sequencing include, e.g., pyrosequencing (such as 454/Roche), polymerase-based sequence-by-synthesis (such as offered by Illumina), ligation-based sequencing (such as offered by SOLiD).

Another possible approach is commercialized by NanoString Technologies. Single mRNA molecules are individually tagged with fluorescent barcodes (“nanostrings”), which can be detected one-by-one and counted for direct digital quantification. The advantage of this approach is that it does not rely on analog quantification of fluorescent intensity, which can be problematic due to noise, lack of linearity, and narrow dynamic range. Instead, the technique relies on fluorescence to detect simply the presence of a single mRNA molecule in a binary (“yes” or “no”) mode. No amplification is necessary, and detection can often be done without much pre-processing of the sample.

Of course, many more platforms is exist, and the disclosure is not limited to a particular methodology. Other examples of technology that could be used for data gathering or analysis include, e.g., the Dynamic Multi-Analyte Technology (“DMAT”) platform of BIOCARTIS®, or qbaseplus (Biogazelle) for automated and improved analysis.

When the gene product to be determined is/are proteins, the expression level can be directly assessed by a number of means with some clear analogies to the techniques for mRNA quantification. Examples include Western blot, ELISA, FACS, two dimensional electrophoresis, mass spectrometry-based methods, isotope-coded affinity-tag-based protein profiling (ICAT), multidimensional protein identification technology (MudPit), peptide arrays, and the like. According to particular embodiments, the signature measures expression of membrane-expressed biomarkers, so that, e.g., FACS can be used to sort and/or determine expression levels.

This list provides a few exemplary technologies, but is by no means exhaustive. The skilled person will be aware of other methods to determine mRNA or protein levels, and these methods are envisaged as well. The nature of the method is not vital to the disclosure, as long as expression of a gene product can be determined.

The methods presented herein involve determining expression of at least one gene product in a sample of monocytes. The expression of this gene product can then be correlated to presence of a disease, i.e., the at least one gene product serves as a marker. Whereas it is possible to use one gene product as marker for a disease, it is, of course, also envisaged to use more than a single marker to make the diagnosis more robust; e.g., a selection of 2 markers may be used, of 3 markers, of 4 markers, 5 markers, 6 markers, 7 markers, 8 markers, 9 markers, or of 10 or even more. For instance, part or the full 29 marker list of Table III can be used as marker panel (i.e., a selection from the following list of genes: ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF. More particularly, a selection from the genes: ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, S100P, SOCS3, TKT, and TNF.). Even more particularly, a selection is made from the genes: ACP5, ADM, ALDH1A1, APP, CD68, CTSZ, DDIT4, FKBP5, GPER, HBB, HP, LAPTM4A, S100P, SOCS3, TAF15 and TKT; even more particularly, a selection is made from the genes: ACP5, ADM, ALDH1A1, APP, CD68, CTSZ, FKBP5, GPER, HBB, HP, LAPTM4A, S100P, SOCS3, TAF15 and TKT. Alternatively, part or the full 43 marker list of Table IV can be used as marker panel. Examples of subsets there include, but are not limited to, a selection from the list of: ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, LOC644063, S100P, SEPT5, SLPI, SOCS3, and TKT. More particularly, a selection of the list of: ADM, APP, BAX, CD68, CTSZ, CXCR4, FKBP5, GPER, HBA1, HBB, LAPTM4A, S100P, SEPT5 and TKT. Genes are indicated herein using their official symbols, as provided by the HUGO Gene Nomenclature Committee (HGNC).

Alternatively, part or the full 45 marker list of Table II can be used as marker panel, part or the full marker list of Table IV, or even part or the full marker list of the 336 probes (genes listed in Table I). These markers are particularly useful for diagnosing the presence and/or the stage of colon cancer. Including extra markers may increase robustness and/or reliability of the diagnosis, or may offer extra information regarding disease progression or stage. The number of markers can in principle be increased ad libitum, although this may be limited for practical reasons (cost, methodology, no more increase in information, etc.).

According to some specific embodiments, it may be useful to exclude particular markers. Most particularly, the at least one gene product whose expression is determined is not a gene product that is used as marker to identify the nature of monocytes in said sample. It can indeed be foreseen that if a marker is used for monocyte isolation, this marker will automatically be enriched in the isolated cells. Thus, increased expression of such marker is not a change that depends on presence of disease, but may be an artifact of the isolation procedure. Therefore, the marker(s) for diagnosing disease are preferably different from that/those used in obtaining or isolating the monocyte population.

Accordingly, as envisaged in these embodiments, the at least one gene product whose expression is determined in the sample or population of monocytes is not a gene product that was used to characterize, isolate or separate the monocytes. Thus, if CD14 was used to establish the monocyte nature of the cells, the at least one gene product whose expression is determined is not a product of the CD14 gene. Likewise, if expression of an additional subset marker, e.g., Tie-2, is used to determine the subpopulation of monocytes, then this additional marker is not a marker whose expression is used to diagnose presence, absence or status of disease. In general terms, particular monocyte markers or monocyte differentiation markers are excluded as markers to determine disease status.

Typically, expression of the at least one gene is altered in the monocytes of a subject with the disease as compared to a healthy control. The altered expression levels of the at least one gene product are then indicative of the disease. Altered gene expression levels may be increased or decreased expression levels, and the increase or decrease in expression levels is then indicative of disease. To increase the detection window, the level of genes that are increased can be divided by the level of genes that are decreased (or vice versa), as this will amplify the difference when expression is altered without affecting the result of this division when gene levels are not both altered in the respective direction. Moreover, whereas a first category of markers presented herein are suited to discriminate between healthy and disease status, a second category of markers is useful to characterize disease severity (i.e., grade the disease). Note that these categories are not mutually exclusive: a marker may be useful both for discriminating between healthy and disease state and for determining disease severity. This is because the expression of marker genes does not necessarily remain stable over time. Indeed, marker genes whose expression is up-regulated in monocytes upon presence of a disease either may retain their increased expression, may further be increased upon progression of the disease, or expression levels may be down-regulated upon disease progression (in the latter case, they may be down-regulated but expression levels are still higher than those found in healthy individuals, they may be down-regulated to the levels found in healthy subjects, or expression levels may be down-regulated to levels that are lower than those found in healthy controls). Depending on the statistical relevance of the expression change (compared to healthy control or, e.g., compared to early stages of disease), the skilled person can decide for which change he uses the markers. The same applies, mutatis mutandis, for marker genes whose expression is down-regulated upon presence of a disease.

A typical example of disease progression can be found in cancer, where in early stages the cancer is present only as a solid tumor, while in later stages the tumor has metastasized. Markers indicative of progression will then be able to distinguish the metastasized state.

By way of non-limiting examples, for colon cancer, expression of DDIT4 or CXCR4 in monocytes is a very relevant marker to discriminate between healthy individuals and patients with cancer, but the marker is less suited to discriminate between people with or without metastasis. FKBP5 and GPER expression levels can be used as a marker to discriminate between healthy people, people with only solid tumors, and subjects with metastasized colon cancer. ENSA expression levels can be used to discriminate patients with a solid tumor from healthy controls as its expression levels are initially increased. In patients with metastasized tumors, the expression levels decrease again (cf. FIG. 2). Thus, if it is known that cancer is present (e.g., by using another marker presented herein), the lower ENSA levels will indicate that the cancer is in an advanced state, as the decrease of ENSA expression compared to patients with a non-metastasized tumor is statistically significant. In other words, if ENSA levels are not significantly changed compared to a healthy control, it is envisaged to determine expression of at least one other marker to be able to derive the presence of the disease; ENSA can then be used as a marker to classify the disease stage. The same applies for other markers where the expression difference with healthy controls is only present in either early or advanced disease stage, but not in the other stage.

The use of particular genes for deter nining stage of a disease will be discussed in more detail in the Examples section.

A non-limiting selection of genes that are particularly suited to determine presence of disease (i.e., expression of these genes in monocytes in both early and late disease stages is significantly different from expression in monocytes from healthy controls) include: ACP5, ADM, CXCR4, DDIT4, DNAJC7, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3 and TKT. According to alternative embodiments, the genes that are particularly suited to determine presence of disease are one or more selected from ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, LOC644063, S100P, SEPT5, SLPI, SOCS3, and TKT, as these are all genes that combine good AUC values with good p values in ROC curves comparing combined P, PM groups with the HV group. Other particular subsets include one or more genes selected from the group of: ACP5, ADM, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, S100P, SLPI, SOCS3, TKT, ALDH1A1, and SEPT5. Alternatively, one or more genes selected from the group of: ADM, APP, BAX, CD68, CTSZ, CXCR4, FKBP5, GPER, HBA1, HBB, LAPTM4A, S100P, SEPT5 and TKT. Yet another alternative is one or more genes selected from the list of: ADM, BAX, CD68, CTSZ, CXCR4, FKBP5, GPER, HBA1, HBB, LAPTM4A, S100P, and TKT.

Selected genes that are particularly suited to discriminate between non-metastasized and metastasized cancer include, but are not limited to: ACP5, ADM, ALDH1A1, APP, ENSA, FKBP5, GPER, HLA-DQA1, LOC644063, LOC723972, RN28S1, S100P, TAF15, TKT and TNF. Most particularly envisaged are markers selected from the list of: ACP5, ALDH1A1, ENSA, LOC644063, LOC723972, TAF15, TKT and TNF; as these markers have a very low p value when comparing expression values in monocytes from patients with non-metastasized versus metastasized tumors. Another selection of genes that are particularly suited for discriminating between non-metastasized and metastasized cancer are markers selected from the list of: ALDH1A1, BAX, LOC644063 and TNF. Most particularly envisaged is the use of ALDH1A1.

A selection of genes that are particularly sensitive and specific markers (i.e., have a high area under curve for a ROC curve) for colon cancer (and are thus particularly suited as single markers) include, but are not limited to: ADM, CXCR4, DDIT4, DNAJC7, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, IL1R2, RN28S1, S100P, SOCS3 and TKT. An alternative selection is one or more genes selected from the list of: ADM, BAX, CD68, CTSZ, CXCR4, FKBP5, GPER, HBA1, HBB, LAPTM4A, S100P, and TKT.

The methods as described herein determine gene expression in a sample of monocytes. This sample of monocytes typically has been obtained from a subject at some time prior to determining the gene expression. As monocytes circulate in the blood, normally a blood sample will be taken from the subject from which sample the monocytes can be isolated. Typically, peripheral blood will be used, as this is the easiest to obtain: it is readily accessible and the procedure is minimally invasive. According to particular embodiments, however, central blood can be used for the blood sample from which the monocytes are isolated, e.g., obtained using a central venous catheter. This may be interesting for patients who are already hospitalized and have such a catheter.

Once a blood sample has been procured, the monocyte sample can be obtained by isolating the monocyte population from the rest of the constituents in the blood. There are many possible methods to isolate the monocyte fraction from a total blood sample, and the nature of the isolation is not vital to the disclosure, as long as it results in a relatively pure population of monocytes (i.e., essentially consisting of monocytes, not other cells). By way of non-limiting example: typically, first peripheral blood mononuclear cells (PBMNCs) are separated from the rest of the blood. This can be done for instance by density gradient centrifugation (e.g., using LYMPHOPREP® from Axis-Shield, FICOLL-PAQUE™ PLUS from GE Healthcare; ACCUSPIN™ System HISTOPAQUE®-1077 from Sigma-Aldrich). Monocytes can be isolated from the PBMC fraction by using marker-assisted selection, e.g., using a MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer's protocol, or alternatively by FACS. Typical markers for monocyte selection include CD14, CD16, Slan (6-Sulfo LacNAc). Note that negative selection is also possible, by labeling the non-monocytes (i.e., T cells, NK cells, B cells, dendritic cells and basophils) in the sample using for instance a selection of antibodies against CD3, CD7, CD15, CD16, CD19, CD56, CD123 and Glycophorin A (e.g., using the monocyte isolation kit II from Miltenyi Biotech, Bergisch Gladbach, Germany). Alternatively, monocytes can be isolated using a plastic adherence protocol, as, e.g., described in Elkord et al., 2005.

A one-step isolation process to obtain monocytes from whole blood can, e.g., be envisaged by using a CD14 antibody, e.g., by coating a plate with CD14 antibody, incubating the plate with whole blood, washing the blood away, whereupon the remaining fraction in the dish will be the monocyte fraction.

Although it is not the most preferred option, it is envisaged that the gene signature can be derived from blood directly. According to these very specific embodiments, the at least one gene of which the expression is determined is present only in monocytes, or the change in expression is much bigger in monocytes than in other blood cells. If that is the case, determining the expression in blood cells will reflect the altered expression in monocytes (i.e., the other cells do not interfere with the expression determining step), and only in that case can the blood sample be regarded as equivalent to a sample of monocytes. As it can be envisaged that gene expression is altered in lymphocytes and granulocytes as well (due to different factors, e.g., a common cold has a high influence on the level of granulocytes), such set-up should be carefully evaluated, as it is not expected to work for all genes in all circumstances. However, in such instances where monocyte extraction from blood is not readily feasible, this can be considered an alternative.

As expression of gene products is a dynamic process, it is important to start the isolation of the monocytes from the blood sample within a timeframe where expression levels are not altered again, in order not to obtain false positive or negative results. In other words, freshly isolated blood should be used for isolation of the monocytes, and the blood should be stored appropriately (2-8° C.) before isolation is started. Particularly, monocyte isolation is started within 6 hours of obtaining the blood sample from the subject, more particularly, the monocyte isolation is started within 5 hours, 4 hours or 3 hours of obtaining the blood sample, most particularly, the monocyte isolation is started within 2 hours, 1 hour, 45 minutes, half an hour, 15 minutes, or immediately after the obtaining of the blood sample. Ideally, the monocyte isolation procedure does not take too long, e.g., not more than 4 hours, 3 hours or 2 hours. Accordingly, the monocyte isolation procedure is finished (=the monocyte population is isolated) within 10 hours of obtaining the blood sample from the subject, more particularly within 9 hours, 8 hours, 7 hours or most particularly within 6 hours, 5 hours or less after obtaining the blood sample.

Once the monocytes have been isolated from the blood, expression of at least one gene product of the monocytes can be determined. This can be done immediately after the isolation of the monocytes. However, it is envisaged that the monocytes are thus prepared that they can be used at a later point in time, e.g., by lysing the monocytes in an RNA lysis buffer (e.g., Buffer RLT, Qiagen) and storing the lysate at −80° C. (or lower) for up to 1 year. Note that these pre-processed (particularly lysed) samples are also considered a sample of monocytes herein: even though these do not contain whole monocytes anymore (as the cells are lysed), they are derived from a pure population of monocytes, so the gene expression is as found in the monocyte population.

Typically, when the at least one gene product whose expression is determined is mRNA, reverse transcription polymerase chain reaction (RT-PCR) is used, allowing very low copy numbers of mRNA to be detected. Briefly, a RNA strand is reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using PCR. However, methods to determine mRNA expression where no amplification is necessary also exist (e.g., nanostring technology).

When the at least one gene product whose expression is determined is protein, this is not amplified as there is no PCR procedure for proteins. However, to detect low abundant proteins, detection with primary and secondary antibodies can be performed to amplify the signal.

Determining expression levels at the mRNA levels is preferred, as it allows easy and reliable quantification. Of course, as the skilled person is well aware of, expression levels may be used as such, or normalized (e.g., against a reference gene or synthetic standards), in line with the routine protocols used in the art.

Thus, methods for diagnosing a disease are provided herein, comprising:

-   -   a) Isolating, from a blood sample of said subject, a monocyte         population;     -   b) Determining in said monocyte population the expression or         levels of expression of at least one gene product;     -   c) Correlating the expression or levels of expression of the at         least one gene product with the presence or absence of the         disease in said subject.

As mentioned, the methods may also entail the prior obtaining of the blood sample, preferably not more than 6 hours prior to the isolation of the monocytes.

The methods provided herein are particularly useful for the diagnosis of cancer.

Gene products that show altered expression levels in monocytes when the subject has a disease are provided herein. Correlating the expression levels with the presence or absence of disease (step c) entails comparison of the expression levels of the at least one gene product determined in step b) with reference values (e.g., those of a healthy control) and based on the difference or similarity of these values conclude whether the subject has the disease. As explained earlier, the correlating step may entail a further step of grading the disease stage or severity; this is based on markers that have altered expression levels in early disease stages (e.g., non-metastasized cancer) as compared to late disease stages (e.g., metastasized cancer).

Distinct advantages of the present methods, apart from their remarkable sensitivity and specificity, are the minimal invasiveness of the methods, the fact that no dietary restrictions are necessary, and the speed of the procedure. Indeed, the protocols from drawing of the blood to comparing the expression values can be completed in less than 12 hours.

The methods described herein are also particularly useful to monitor evolution of the disease. This is because the gene signature retains plasticity. Monocytes will show an altered gene expression pattern quite rapidly (within hours rather than days) when exposed to an inflammatory stimulus in the body (e.g., the presence of cancer, such as colorectal cancer. This will happen even if the tumor is very small). However, when the stimulus is taken away (e.g., by surgically resecting the tumor), the monocytes will revert their gene expression to baseline values, and vice versa when the stimulus is introduced again.

Thus, according to particular embodiments, the methods provided herein are used for monitoring recurrence of a cancer. In other words, the subject from whom the monocytes are obtained is a subject that had cancer, but in which the cancer has been removed. The methods are then performed to evaluate whether the treatment of the cancer has been successful, or whether the tumor has returned. This is particularly envisaged for monitoring subjects who are at high risk of recurrence (for example, patients who had poorly differentiated tumors or venous or lymphatic invasion).

According to alternative, but not exclusive, particular embodiments, the methods are used for monitoring response to therapy. For example, once the subject has been diagnosed with cancer (using the methods described herein or alternative methods known in the art), and treatment for cancer has initiated, the present methods are performed again over time, to see whether the monocyte gene expression returns to baseline values (i.e., levels comparable to those found in healthy volunteers), as this would be indicative of a successful treatment and eradication of the tumor. It is envisaged that the present methods are particularly suited in determining minimal residual disease (MRD). MRD is the name given to small numbers of cancer cells that remain in the patient during treatment, or after treatment when the patient is in remission (no symptoms or signs of disease). In cancer treatment, MRD testing has several important roles: determining whether treatment has eradicated the cancer or whether traces remain, comparing the efficacy of different treatments, monitoring patient remission status and recurrence of the cancer and choosing the treatment that will best meet those needs (personalization of treatment). Given the fact that even very small tumors are detected with the present gene signature, it is particularly envisaged that the signature presented herein is highly suitable for MRD testing even when the patient has no other symptoms or signs of disease.

As the altered gene expression correlates to the presence of disease and can be used to monitor response to therapy, it is also envisaged that the methods provided herein may comprise an additional step of changing the treatment of the patient based on the results of the diagnosis. For instance, if the patient is receiving a combination of chemotherapeutics, but this does not result in gene expression in the monocytes reverting back to baseline values, this is indicative that the therapy does not work, and a decision may be taken to switch to a different chemotherapy regimen (e.g., different or additional drugs, or different doses of the same drugs).

Many different treatment options exist for colorectal cancer, by way of non-limiting examples, these include one or more of the following: local excision (for Stage 0 or Stage I tumors), radiotherapy, chemotherapy (e.g., with SFU, the FOLFOX regimen (oxaliplatin, 5-FU, and leucovorin), 5-FU and leucovorin, CapeOx (capecitabine plus oxaliplatin) or capecitabine alone, FOLFIRI (leucovorin, 5-FU, and irinotecan), FOLFOXIRI (leucovorin, 5-FU, oxaliplatin, and irinotecan), bevacizumab, cetuximab, irinotecan, panitumumab), and chemoradiation (combination of chemotherapy and radiation).

According to a further aspect of the disclosure, a gene signature is provided. This gene signature typically comprises at least two genes, wherein at least one gene is selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF; and at least one other gene is selected from the genes listed in Table I, II, III and/or IV. Alternatively, at least one gene is selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, LOC644063, S100P, SEPT5, SLPI, SOCS3, and TKT; and at least one other gene is selected from the genes listed in Tables I, II, III and/or IV. Both the “at least one” gene, and the “at least one” other gene may, according to particular embodiments, and independent of each other, also be at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten.

According to further particular embodiments, at least two genes from the gene signature are selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF (or from the alternative list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, LOC644063, S100P, SEPT5, SLPI, SOCS3, and TKT). Since these genes are also present in Tables I, II, III and/or IV, this obviates the need for an additional gene from these tables (although further genes may still be added). According to yet further particular embodiments, at least 3, 4, 5, 6, 7 or 8 genes from the gene signature are selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF (or from the alternative list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, LOC644063, S100P, SEPT5, SLPI, SOCS3, and TKT).

Although the gene signature contains at least 2 genes, in practice the signature may contain more genes, e.g., at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes. When cost or technology is not limiting the number of genes the expression of which is determined, the gene signature may even contain at least 10 genes, at least 12 genes, at least 16 genes or at least 20 genes.

However, as it is envisaged that only up to a certain amount the gene expression will add extra information (e.g., confidence in prediction: added sensitivity or specificity), it is also envisaged that the gene signature contains no more than 50 genes, or no more than 40 genes, no more than 30 genes, no more than 25 genes, no more than 20 genes, no more than 16 genes, or no more than 12 genes. In this regard, it should be mentioned that some genes show co-regulation of expression and can be substituted for one another in the analysis. For instance, the information contained in the expression of CXCR4 correlates highly with that of TKT, and the information contained in the expression of HBA1 is highly correlated to that in HBB, so inclusion of both genes of such a gene pair may be redundant in a signature set.

Of course, the upper and lower limits described herein can be combined. Accordingly, typical gene signatures may comprise, e.g., between 2 and 20 genes, between 2 and 16 genes, between 3 and 16 genes, between 4 and 16 genes, between 3 and 12 genes, between 4 and 12 genes, etc. (with always at least one selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF; and the others from that preferred list or from Tables I, II, III, and/or IV). According to alternative embodiments, at least one gene is selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, LOC644063, S100P, SEPT5, SLPI, SOCS3, and TKT; and the others from that preferred list or from Tables I, II, III, and/or IV.

According to yet further particular embodiments, at least one gene is selected from the list of ADM, BAX, CD68, CTSZ, CXCR4, FKBP5, GPER, HBA1, HBB, LAPTM4A, S100P, and TKT, and optional others are selected from that same list, or from Tables I, II, III and/or IV.

According to very specific embodiments, the gene signature contains at least one gene whose expression is down-regulated in a disease setting, while the expression of at least one gene is up-regulated in this disease setting.

According to most specific embodiments, the gene signature is a monocyte gene signature, i.e., a group of genes with altered expression in monocytes, characteristic for a particular condition (particular cancer, most particular colorectal cancer).

Of note, although ideally all selected markers will show altered expression in patients (or metastatic patients) as compared to healthy controls, in practice not all markers will always reach significantly different expression levels (or the outcome is unsure). This is another reason why it is beneficial to assess expression of more than one marker. In such instances, it is envisaged that a sample is classified as positive (e.g., as indicative of the presence of disease) if at least a certain percentage of markers shows significantly different expression (higher or lower, depending on the marker). This may for instance be at least 50% of markers, at least 60% of markers, at least 70% of markers, at least 75% of markers, at least 80% of markers, at least 90% of markers, or even at least 95% of markers. Typically, the higher the number of genes whose expression is assessed, the lower the percentage of markers that needs to be positive to allow correlation with disease. For instance, when all 43 markers of Table IV are used, and 50% of them show significantly altered expression, this will be indicative of the presence of disease. On the other hand, when a panel of 16 (or 12) markers is used, it is typically envisaged that at least 70% (or even at least 75%) of them needs to show a significantly altered expression to correlate this to the presence of disease.

The gene signature is particularly useful in the field of medicine. Thus, according to specific embodiments, the gene signature provided herein is provided for use as a medicament. Particularly, it is provided for use as a diagnostic. More particularly, the gene signature provided herein is provided for use in the diagnosis of cancer. Most particularly, the gene signature provided herein is provided for use in the diagnosis of colorectal cancer.

According to a further aspect, kits are provided comprising means to detect the gene signature. For example, kits are provided that contain means to detect expression or levels of expression of at least two genes, wherein at least one gene is selected from the list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SOCS3, TAF15, TKT, and TNF (or the alternative list of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, LOC644063, S100P, SEPT5, SLPI, SOCS3, and TKT); and at least one other gene is selected from the genes listed in Tables I, II, III and/or IV.

It is envisaged that these kits are provided for a particular purpose, such as for diagnosis of cancer, or more particularly, for the diagnosis of colorectal cancer.

The means to detect expression and/or expression levels of the at least two genes that are provided in the kits will depend on the methodology used to detect expression. As mentioned, detection can be on nucleic acid or protein level. For protein-based detection, the kits typically will contain antibodies for the at least two genes whose expression will be determined. Further control antibodies (e.g., against housekeeping genes) may also be provided in the kit.

Likewise, for detection at the nucleic acid level, the kits will typically contain primers or probes for the at least two genes whose expression will be determined. Further control primers and probes (e.g., against housekeeping genes) may also be provided in the kit.

The kit may further also contain standards or controls to check whether determining the expression has been done successfully (e.g., samples with known concentration of the at least two genes whose expression is determined, to compare the obtained values, or to check whether the detection reaction worked).

Of course, the kit may further comprise pharmaceutically acceptable excipients, buffers, and an instruction manual and so on.

It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the disclosure, various changes or modifications in form and detail may be made without departing from the scope and spirit of this disclosure. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

Examples Example 1 Identification of Predictive Biomarkers by Gene Profiling of PBM from CRC Patients

Three groups of untreated participants were enrolled to evaluate gene expression in monocytes. The first group included 27 newly diagnosed colorectal cancer patients with no sign of detectable metastasis (abbreviated as P). The second group included 28 cancer patients with clinically evident metastasis at the time of diagnosis (indicated as PM). The third group included 38 age and gender-matched healthy volunteers (HV). Venous peripheral blood (20 ml) was collected at the time of first diagnosis. Clinical-pathological characteristics of participants were determined at diagnosis such as gender, age, tumor localization, histological tumor grading, and tumor stages according to the TNM and International Union against Cancer (UICC) classifications. The exclusion criteria were the following: <45 and >75 years, previous history of oncological, inflammatory or autoimmune diseases, anti-inflammatory therapy, i.e., corticosteroids and NSAID at the time point of enrollment. Only Belgian cancer patients and healthy volunteers were enrolled in this monocentric study to reduce ethnical and environmental variability.

Isolation of monocytes from patients' peripheral blood was done using a two-step method: peripheral blood mononuclear cells (PBMNCs) were collected from fresh heparinized blood using a Ficoll density gradient and monocytes were isolated from PBMNCs by magnetic separation using magnetic microbeads conjugated to antibodies against CD14, a specific marker for human monocytes. Part of the PBM collected were used directly to study the gene expression profile while part of them were frozen to perform in vitro analysis (see further).

RNA was extracted from isolated monocytes and gene expression profiles of PBM were investigated by high-throughput screening using Illumina Bead Array platforms. Using statistical analysis of microarray expression data, a disease-specific genetic signature of maximum specificity and sensitivity was delineated, exploiting state-of-the art disease classification algorithms in biomarker development. The gene expression data was analyzed based on the Limma package from Bioconductor. At first, three groups were compared: metastatic patients (PM) versus healthy volunteers (HV), non-metastatic patients (P) versus HV, and PM versus P. In a separate additional analysis, the distinction between PM and P was omitted and this combined group, i.e., all patients with colorectal cancer, was compared towards HV.

1.1 Statistical Analysis of the Microarray Data

After quality assessment and data normalization on the 93 samples, differentially expressed genes between the groups can be assessed.

1.1.1 Data Filtering

The total number of probes on the array is 47,323. For each probe, we count the number of present detection calls, over all samples. This ranges between 0 (none are present) and 93 (all are present). We omit the probes that were absent for all samples (i.e., number of present calls=0) from the analysis. This subset consists out of 18,396 probes. Hence analysis is performed for 28,927 probes.

1.1.2 Assessing Differentially Expressed Probes

We compare the normalized expression values of the different conditions with the limma package of Bioconductor (Smyth, 2004; Smyth, 2005). We estimate an average expression value for the three groups:

-   -   1. HV (#=38 samples)     -   2. PM (#=28 samples)     -   3. P (#=27 samples)

Based on these estimates, we can estimate the contrasts of interest. In a second similar analysis, we compute the estimates for the combined group of PM and P (#=55 samples) and we estimate the contrast of (PM, P) versus HV. For each of the contrasts, we test whether it is significantly deviating from 0 with a moderated t-statistic (implemented in limma). The resulting p-values are corrected for multiple testing with Benjamini-Hochberg (Benjamini and Hochberg, 1995) to control the false discovery rate.

Different selection methods can be applied for the selection of differentially expressed genes. One can select based on the p-values, corrected for multiple testing, e.g., all probes with a corrected p-value less than 0.05. Or, one can apply a more stringent cut-off of the uncorrected p-values, e.g., p<0.001. A cut off on the p-values can be combined with a cut-off on the fold-change. By way of example, combining a cutoff of p<0.05 after multiple testing correction according to Benjamini-Hochberg with a cut-off set to only select the genes showing a minimum fold change of 1.5 (log-ratios to 0.58) yields the following numbers of differentially expressed genes.

p < 0.05 Comparison Log-ratio <−0.58 Log-ratio >0.58 PM versus HV 4 29 P versus HV 5 26 PM versus P 0 0 (PM, P) versus HV 4 36 (negative log ratios indicate genes that are down-regulated, positive log ratios are genes that are up-regulated).

The analysis showed that there was a broad overlap in the gene signature of PBM from non-metastatic and metastatic patients, indicating that this signature already exists in the early stages of CRC and remains stable during the progression towards metastatic CRC. Based on this, we decided to assess the performance of the gene signature of (PM, P) versus HV as diagnostic tool.

1.2 Construction of Gene Signatures

For most datasets, the limma method is a very reliable approach for constructing a gene signature. However, to optimize the limma-signature even further, alternative signatures are constructed, evaluated with classification approaches and finally merged with the limma signature. Seven different approaches to construct gene signatures were used. Five of them are combinations of three ranking methods, followed by one of two feature selection modes.

The three ranking methods are based on limma (Smyth, 2004), DEDS (Hwa and Yuanyuan, 2005) or PCA. The limma-ranking ranks genes according to uncorrected p-values. With the DEDS package, we rank genes based on several criteria simultaneously (including fold changes, ordinary t-statistics, ordinary F-statistics, moderated t-statistics, moderated F-statistics, SAM (significance analysis for microarrays) statistics and B-statistics). We use simple Euclidean distance to evaluate contributions from different statistics. PCA-ranking starts with principal component analysis. We evaluate every component with a t-test for its capacity to distinguish between sample classes and retain the five components with smallest p-values. To each probe set, a weight is attributed in accordance to its contribution in such a principal component (Eijssen et al., 2008). Weights from several components are combined by a weighted sum, with weights in accordance to the singular value of the component. The weights are used for the so called PCA-ranking. The two modes of feature selection that we use after ranking are filtering or wrapping. With filtering, we use a fixed criterion to decide which top genes from the ranking we will select. For filtering after limma-ranking, we use a stringent cut-off on the p-values of the moderated t-statistic, e.g., p<0.001, combined with a cut-off on fold-change of two (i.e., an absolute log 2-ratio larger than 1). For filtering after DEDS-ranking, we select only differentially expressed genes with a p-value <0.05. For filtering after PCA-ranking, we select only the 5*nsamps top genes from the ranking, with nsamps equal to the number of samples in the dataset. With wrapping the top-1 to top-nsamps are step-wise presented for further classification (see below) (Daemen et al., 2009).

Finally, two approaches to construct gene signatures use an embedded filtering mode (Saeys et al., 2007). No ranking is performed: nearest shrunken centroid and random forests (see below) have an internal procedure for direct feature selection.

1.2.1 Evaluation of Gene Signatures

Gene signatures are evaluated by their predictive power, i.e., how good can we discriminate between the two sample classes when limiting our view only to the expression of the genes in the signature. To determine the predictive power, we compare five different classification algorithms (Statnikov et al., 2008) since no single algorithm outperforms the others on every dataset: k-Nearest-Neighbor (kNN) (Cover and Hart, 1967), support vector machine (SVM) (Burges, 1998), nearest shrunken centroid (NSC) (Tibshirani et al., 2002), random forest (RF) (Diaz-Uriarte and Alvarez de Andres, 2006), and ensemble method (Dietterich, 2000). All together, we consider 24 classification approaches, which are specific combinations of classification algorithms, feature selection modes and ranking methods.

As training and evaluation framework we use Monte Carlo cross-validation (MCCV) with an outer and inner loop (Wessels et al., 2005).

To create balanced datasets, in each cycle excess samples are randomly removed from the training data.

The predictive performance of a classification approach is measured by its balanced error rate (BER). When several approaches have the same optimal BER, we check which one has the lowest area under the ROC curve (AUC) (Bradley, 1997). We select the approach with the lowest BER (or highest AUC) as the optimal approach. To compute the BER, we need the discrete class predictions. For the AUC, we use the continuous prediction values of the approaches.

After a MCCV run, we can compute for each of the 24 classification approaches a ROC-curve that gives a visual overview of the trade-off between sensitivity and specificity. A selection of these curves is shown in FIG. 1. It can be seen that many of these approaches not only perform quite well, but also have similar ROC curves.

1.2.2 Optimization of Gene Signature for Functional Analysis

After the evaluation, we select the optimal classification approach and two “second-best” approaches that have alternative gene signatures as input. Here, we opted for NSC_ALL_embedded as optimal approach. As “second-best” approaches with alternative signatures, we take SVM_LIMMA_wrapper and SVM_DEDS_wrapper. These three approaches are then trained on the complete dataset. However, training should best be conducted on a balanced dataset (equal number of samples in each class). Therefore, as during the evaluation scheme, a random set of samples is discarded to make the dataset balanced. To compensate for the random element that is introduced by this procedure, we repeat the balance-correcting scheme 25 times, each time discarding another random set of samples. In this way, we compute for each gene the reproducibility score, i.e., the percentage of repeats in which this probe was selected for the gene signature. The three balance-corrected signatures are then identified as the sets consisting of genes with a reproducibility score >75.

Alternative methods for the selection of gene signatures often select different gene sets from the same pathways (Shi et al., 2010; Statnikov and Aliferis, 2010). Hence, in order to create gene signatures with optimal stability/reproducibility and biological interpretability, alternative, good signatures may be merged into one final signature. Therefore, we assemble the final gene signature as the union of the limma-signature and the balance-corrected signatures of the optimal classifier and two second-best classifiers. The final, merged signature contains 336 probes. The list of probes and corresponding target genes is shown in Table I. Based on balanced error rates, we estimate that a SVM-classifier with the signature as input would classify correctly about 93% of cases (in a balanced setup).

TABLE I List of target genes identified by Illumina analysis, together with an indication whether they are up-regulated (positive log ratio or “up”) or down-regulated (negative log ratio or “down”). Log ratio PMP vs. HV Target ID ACCESSION DEFINITION 1.425282722 HBA2 NM_000517.3 Homo sapiens hemoglobin, alpha 2 (SEQ ID NO: 1) (HBA2), mRNA. 1.287783085 HBA2 NM_000517.3 Homo sapiens hemoglobin, alpha 2 (SEQ ID NO: 1) (HBA2), mRNA. 1.256987293 HBB NM_000518.4 Homo sapiens hemoglobin, beta (HBB), (SEQ ID NO: 2) mRNA. 1.222508533 HBA1 NM_000558.3 Homo sapiens hemoglobin, alpha 1 (SEQ ID NO: 3) (HBA1), mRNA. 0.998509041 ADM NM_001124.1 Homo sapiens adrenomedullin (ADM), (SEQ ID NO: 4) mRNA. 0.991062267 TAF15 NM_139215.1 Homo sapiens TAF15 RNA polymerase (SEQ ID NO: 5) II, TATA box binding protein (TBP)- associated factor, 68 kDa (TAF15), transcript variant 1, mRNA. 0.836689364 CTSZ NM_001336.2 Homo sapiens cathepsin Z (CTSZ), (SEQ ID NO: 6) mRNA. 0.819643571 TAF15 NM_003487.2 Homo sapiens TAF15 RNA polymerase (SEQ ID NO: 7) II, TATA box binding protein (TBP)- associated factor, 68 kDa (TAF15), transcript variant 2, mRNA. 0.777181029 DDIT4 NM_019058.2 Homo sapiens DNA-damage-inducible (SEQ ID NO: 8) transcript 4 (DDIT4), mRNA. 0.725481681 LOC100132394 XM_001713809.1 PREDICTED: Homo sapiens (SEQ ID NO: 9) hypothetical protein LOC100132394 (LOC100132394), mRNA. 0.688401645 LOC153561 NM_207331.2 Homo sapiens hypothetical protein (SEQ ID NO: 10) LOC153561 (LOC153561), mRNA. 0.682952046 FKBP5 NM_004117.2 Homo sapiens FK506 binding protein 5 (SEQ ID NO: 11) (FKBP5), mRNA. 0.682258483 HS.61208 BF434110 7o98h02.x1 NCI_CGAP_Ov18 Homo (SEQ ID NO: 12) sapiens cDNA clone IMAGE: 3644403 3, mRNA sequence 0.672157697 DNAJC7 NM_003315.1 Homo sapiens DnaJ (Hsp40) homolog, (SEQ ID NO: 13) subfamily C, member 7 (DNAJC7), mRNA. 0.662987224 GPER NM_001039966.1 Homo sapiens G protein-coupled (SEQ ID NO: 14) estrogen receptor 1 (GPER), transcript variant 3, mRNA. 0.646983223 SEPT5 NM_002688.4 Homo sapiens septin 5 (SEPT5), (SEQ ID NO: 15) mRNA. 0.645562444 TKT NM_001064.1 Homo sapiens transketolase (Wernicke- (SEQ ID NO: 16) Korsakoff syndrome) (TKT), mRNA. 0.644703597 LOC649143 XM_944822.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 17) HLA class II histocompatibility antigen, DRB1-9 beta chain precursor (MHC class I antigen DRB1*9) (DR-9) (DR9), transcript variant 2 (LOC649143), mRNA. 0.629326367 LOC100008589 NR_003287.1 Homo sapiens 28S ribosomal RNA (SEQ ID NO: 18) (LOC100008589), non-coding RNA. 0.626313819 HP NM_005143.2 Homo sapiens haptoglobin (HP), (SEQ ID NO: 19) mRNA. 0.626113556 GPER NM_001039966.1 Homo sapiens G protein-coupled (SEQ ID NO: 14) estrogen receptor 1 (GPER), transcript variant 3, mRNA. 0.625858859 LOC100170939 NR_024054.1 Homo sapiens glucuronidase, beta (SEQ ID NO: 20) pseudogene (LOC100170939), non- coding RNA. 0.618893497 HMOX1 NM_002133.1 Homo sapiens heme oxygenase (SEQ ID NO: 21) (decycling) 1 (HMOX1), mRNA. 0.616165331 S100P NM_005980.2 Homo sapiens S100 calcium binding (SEQ ID NO: 22) protein P (S100P), mRNA. 0.61127259 LOC100134364 XM_001713810.1 PREDICTED: Homo sapiens (SEQ ID NO: 23) hypothetical protein LOC100134364 (LOC100134364), mRNA. 0.604580071 SLC39A1 NM_014437.3 Homo sapiens solute carrier family 39 (SEQ ID NO: 24) (zinc transporter), member 1 (SLC39A1), mRNA. 0.597605882 HS.143909 BX106581 BX106581 Soares_testis_NHT Homo (SEQ ID NO: 25) sapiens cDNA clone IMAGp998E204492, mRNA sequence 0.593231994 SOCS3 NM_003955.3 Homo sapiens suppressor of cytokine (SEQ ID NO: 26) signaling 3 (SOCS3), mRNA. 0.582007674 BAX NM_138765.2 Homo sapiens BCL2-associated X (SEQ ID NO: 27) protein (BAX), transcript variant sigma, mRNA. 0.567951821 HS.581828 BG207842 RST27329 Athersys RAGE Library (SEQ ID NO: 28) Homo sapiens cDNA, mRNA sequence 0.560181209 LOC723972 NR_003144.1 Homo sapiens hepatopoietin PCn127 (SEQ ID NO: 29) (LOC723972), non-coding RNA. 0.558747252 LOC100190986 NR_024456.1 Homo sapiens hypothetical (SEQ ID NO: 30) LOC100190986 (LOC100190986), non- coding RNA. 0.556694791 UPP1 NM_003364.2 Homo sapiens uridine phosphorylase 1 (SEQ ID NO: 31) (UPP1), transcript variant 1, mRNA. 0.555850341 AQP9 NM_020980.2 Homo sapiens aquaporin 9 (AQP9), (SEQ ID NO: 32) mRNA. 0.545469818 TNPO1 NM_153188.2 Homo sapiens transportin 1 (TNPO1), (SEQ ID NO: 33) transcript variant 2, mRNA. 0.53910369 BRD3 NM_007371.2 Homo sapiens bromodomain containing (SEQ ID NO: 34) 3 (BRD3), mRNA. 0.533994393 SLC2A3 NM_006931.1 Homo sapiens solute carrier family 2 (SEQ ID NO: 35) (facilitated glucose transporter), member 3 (SLC2A3), mRNA. 0.532202222 ACP2 NM_001610.1 Homo sapiens acid phosphatase 2, (SEQ ID NO: 36) lysosomal (ACP2), mRNA. 0.515055033 TMBIM1 NM_022152.4 Homo sapiens transmembrane BAX (SEQ ID NO: 37) inhibitor motif containing 1 (TMBIM1), mRNA. 0.506880701 RNF146 NM_030963.2 Homo sapiens ring finger protein 146 (SEQ ID NO: 38) (RNF146), mRNA. 0.495913441 CHD8 NM_020920.2 Homo sapiens chromodomain helicase (SEQ ID NO: 39) DNA binding protein 8 (CHD8), mRNA. 0.490191625 PIK3R1 NM_181523.1 Homo sapiens phosphoinositide-3- (SEQ ID NO: 40) kinase, regulatory subunit 1 (alpha) (PIK3R1), transcript variant 1, mRNA. 0.483060794 HS.313056 BM974067 UI-CF-EC1-abz-d-11-0-UI.s1 UI-CF- (SEQ ID NO: 41) EC1 Homo sapiens cDNA clone UI-CF- EC1-abz-d-11-0-UI 3, mRNA sequence 0.479167584 LOC440345 XM_933717.1 PREDICTED: Homo sapiens (SEQ ID NO: 42) hypothetical protein LOC440345, transcript variant 6 (LOC440345), mRNA. 0.473451156 FCGR1A NM_000566.2 Homo sapiens Fc fragment of IgG, high (SEQ ID NO: 43) affinity Ia, receptor (CD64) (FCGR1A), mRNA. 0.473233358 HS.565917 AW451125 UI-H-BI3-alg-g-11-0-UI.s1 (SEQ ID NO: 44) NCI_CGAP_Sub5 Homo sapiens cDNA clone IMAGE: 2736956 3, mRNA sequence 0.472244147 LOC646103 XM_377879.3 PREDICTED: Homo sapiens similar to (SEQ ID NO: 45) TBP-associated factor 11 (LOC646103), mRNA. 0.471640717 HS.545048 AL042883 DKFZp434I1922_s1 434 (synonym: (SEQ ID NO: 46) htes3) Homo sapiens cDNA clone DKFZp434I1922 3, mRNA sequence 0.469623319 CLEC4D NM_080387.4 Homo sapiens C-type lectin domain (SEQ ID NO: 47) family 4, member D (CLEC4D), mRNA. 0.466971624 LOC728069 XM_001128421.1 PREDICTED: Homo sapiens (SEQ ID NO: 48) hypothetical LOC728069 (LOC728069), mRNA. 0.45205922 HS.543412 AW237129 xm65h05.x1 NCI_CGAP_GC6 Homo (SEQ ID NO: 49) sapiens cDNA clone IMAGE: 2689113 3, mRNA sequence 0.451555789 HS.291195 BX105310 BX105310 Soares_testis_NHT Homo (SEQ ID NO: 50) sapiens cDNA clone IMAGp998J212640, mRNA sequence 0.448701357 SDHC NM_003001.2 Homo sapiens succinate dehydrogenase (SEQ ID NO: 51) complex, subunit C, integral membrane protein, 15 kDa (SDHC), nuclear gene encoding mitochondrial protein, mRNA. 0.448161441 MCOLN1 NM_020533.1 Homo sapiens mucolipin 1 (MCOLN1), (SEQ ID NO: 52) mRNA. 0.443476719 LOC642771 XM_926193.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 53) Golgi autoantigen, golgin subfamily A member 2 (Golgi matrix protein GM130) (LOC642771), mRNA. 0.442898675 ZNF33A NM_006974.2 Homo sapiens zinc finger protein 33A (SEQ ID NO: 54) (ZNF33A), transcript variant 2, mRNA. 0.435332776 HS.552008 CA429430 UI-H-FH1-bfm-b-13-0-UI.s1 (SEQ ID NO: 55) NCI_CGAP_FH1 Homo sapiens cDNA clone UI-H-FH1-bfm-b-13-0-UI 3, mRNA sequence 0.435041868 DYSF NM_003494.2 Homo sapiens dysferlin, limb girdle (SEQ ID NO: 56) muscular dystrophy 2B (autosomal recessive) (DYSF), mRNA. 0.428415348 HS.121635 AW173314 xj85h08.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 57) Homo sapiens cDNA clone IMAGE: 2664063 3 similar to contains TAR1.t2 TAR1 repetitive element;, mRNA sequence 0.425975261 HS.571207 BX119561 BX119561 NCI_CGAP_Kid11 Homo (SEQ ID NO: 58) sapiens cDNA clone IMAGp998K155924; IMAGE: 2385542, mRNA sequence 0.425348595 HS.564676 AW298401 UI-H-BW0-ajj-h-10-0-UI.s1 (SEQ ID NO: 59) NCI_CGAP_Sub6 Homo sapiens cDNA clone IMAGE: 2732035 3, mRNA sequence 0.419931568 HS.539119 DB337375 DB337375 TESTI2 Homo sapiens (SEQ ID NO: 60) cDNA clone TESTI2020522 3, mRNA sequence 0.419918039 HS.582550 BG208291 RST27783 Athersys RAGE Library (SEQ ID NO: 61) Homo sapiens cDNA, mRNA sequence 0.41773203 HS.565086 BF512455 UI-H-BI3-alw-d-07-0-UI.s1 (SEQ ID NO: 62) NCI_CGAP_Sub5 Homo sapiens cDNA clone IMAGE: 3068964 3, mRNA sequence 0.415707991 HS.582136 CN288601 17000583181795 GRN_PRENEU (SEQ ID NO: 63) Homo sapiens cDNA 5, mRNA sequence 0.414728151 AIF1L NM_031426.2 Homo sapiens allograft inflammatory (SEQ ID NO: 64) factor 1-like (AIF1L), transcript variant 1, mRNA. 0.40310815 HS.126573 BX109006 BX109006 NCI_CGAP_Lu5 Homo (SEQ ID NO: 65) sapiens cDNA clone IMAGp998C013947, mRNA sequence 0.402219324 HS.571035 BC033504 Homo sapiens, clone IMAGE: 5168221, (SEQ ID NO: 66) mRNA 0.401439862 CST7 NM_003650.2 Homo sapiens cystatin F (leukocystatin) (SEQ ID NO: 67) (CST7), mRNA. 0.400934362 IER3 NM_003897.3 Homo sapiens immediate early response (SEQ ID NO: 68) 3 (IER3), mRNA. 0.399831112 PLIN2 NM_001122.2 Homo sapiens perilipin 2 (PLIN2), (SEQ ID NO: 69) mRNA. 0.398348917 LOC642981 XM_927840.1 PREDICTED: Homo sapiens (SEQ ID NO: 70) hypothetical protein LOC642981 (LOC642981), mRNA. 0.39472666 HS.543617 AI004472 ot56f09.s1 Soares_testis_NHT Homo (SEQ ID NO: 71) sapiens cDNA clone IMAGE: 1620809 3, mRNA sequence 0.394580918 HS.210969 AI827892 wf12a11.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 72) Homo sapiens cDNA clone IMAGE: 2350364 3, mRNA sequence 0.3945452 ARHGAP26 NM_015071.3 Homo sapiens Rho GTPase activating (SEQ ID NO: 73) protein 26 (ARHGAP26), mRNA. 0.393515945 HNRNPA3 NM_194247.2 Homo sapiens heterogeneous nuclear (SEQ ID NO: 74) ribonucleoprotein A3 (HNRNPA3), mRNA. 0.391976411 HS.563406 AI807179 wf25f10.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 75) Homo sapiens cDNA clone IMAGE: 2356651 3, mRNA sequence 0.386122147 HS.548213 AI630987 tx54c03.x1 NCI_CGAP_Lu24 Homo (SEQ ID NO: 76) sapiens cDNA clone IMAGE: 2273380 3, mRNA sequence 0.38483405 HS.566751 BU753911 UI-1-BC1p-ali-e-01-0-UI.s1 (SEQ ID NO: 77) NCI_CGAP_P13 Homo sapiens cDNA clone UI-1-BC1p-ali-e-01-0-UI 3, mRNA sequence 0.381909307 HS.582025 AI066603 ov47h04.x1 Soares_testis_NHT Homo (SEQ ID NO: 78) sapiens cDNA clone IMAGE: 1640503 3, mRNA sequence 0.381489485 C15ORF28 XR_001373.1 PREDICTED: Homo sapiens (SEQ ID NO: 79) chromosome 15 open reading frame 28 (C15orf28), misc RNA. 0.378073188 HS.562624 BF445990 7p17e02.x1 NCI_CGAP_Br22 Homo (SEQ ID NO: 80) sapiens cDNA clone IMAGE: 3646155 3, mRNA sequence 0.377535176 HS.544451 BE646244 7e83f02.x1 NCI_CGAP_Pr28 Homo (SEQ ID NO: 81) sapiens cDNA clone IMAGE: 3289083 3, mRNA sequence 0.376981852 LOC100132106 XM_001720398.1 PREDICTED: Homo sapiens (SEQ ID NO: 82) hypothetical protein LOC100132106 (LOC100132106), mRNA. 0.373731718 HS.565708 AI961498 wt23e07.x1 NCI_CGAP_Ut1 Homo (SEQ ID NO: 83) sapiens cDNA clone IMAGE: 2508324 3, mRNA sequence 0.371608825 VWA5A NM_014622.4 Homo sapiens von Willebrand factor A (SEQ ID NO: 84) domain containing 5A (VWA5A), transcript variant 1, mRNA. 0.367860736 HS.535906 BF336512 RC2-CT0522-150900-012-g04 CT0522 (SEQ ID NO: 85) Homo sapiens cDNA, mRNA sequence 0.367366826 HS.551330 BE855955 7f85c09.x1 NCI_CGAP_Pr28 Homo (SEQ ID NO: 86) sapiens cDNA clone IMAGE: 3303760 3, mRNA sequence 0.365391324 LOC150568 NR_015399.1 Homo sapiens hypothetical LOC150568 (SEQ ID NO: 87) (LOC150568), non-coding RNA. 0.360112404 SLC35E3 NM_018656.2 Homo sapiens solute carrier family 35, (SEQ ID NO: 88) member E3 (SLC35E3), mRNA. 0.35682846 HS.555208 BM673749 UI-E-EJ0-ahh-i-01-0-UI.s1 UI-E-EJ0 (SEQ ID NO: 89) Homo sapiens cDNA clone UI-E-EJ0- ahh-i-01-0-UI 3, mRNA sequence 0.355509912 HS.545044 CD101933 AGENCOURT_13980472 (SEQ ID NO: 90) NIH_MGC_187 Homo sapiens cDNA clone IMAGE: 30373500 5, mRNA sequence 0.354656089 KCNQ1OT1 NR_002728.2 Homo sapiens KCNQ1 overlapping (SEQ ID NO: 91) transcript 1 (non-protein coding) (KCNQ1OT1), non-coding RNA. 0.354642051 MYST3 NM_006766.2 Homo sapiens MYST histone (SEQ ID NO: 92) acetyltransferase (monocytic leukemia) 3 (MYST3), mRNA. 0.351655619 CCDC135 NM_032269.4 Homo sapiens coiled-coil domain (SEQ ID NO: 93) containing 135 (CCDC135), mRNA. 0.351180052 HIST1H1C NM_005319.3 Homo sapiens histone cluster 1, H1c (SEQ ID NO: 94) (HIST1H1C), mRNA. 0.349986275 LOC653158 XM_926814.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 95) hypothetical protein MGC40405, transcript variant 1 (LOC653158), mRNA. 0.348685879 HS.543863 AW297072 UI-H-BI2-aie-a-06-0-UI.s1 (SEQ ID NO: 96) NCI_CGAP_Sub4 Homo sapiens cDNA clone IMAGE: 2728978 3, mRNA sequence 0.348167026 HS.35496 BX095709 BX095709 Soares pineal gland (SEQ ID NO: 97) 3NbHPG Homo sapiens cDNA clone IMAGp998I24464, mRNA sequence 0.348125204 HS.575808 CF552427 AGENCOURT_15595604 (SEQ ID NO: 98) NIH_MGC_183 Homo sapiens cDNA clone IMAGE: 30530228 5, mRNA sequence 0.347800469 HS.545251 BG192152 RST11259 Athersys RAGE Library (SEQ ID NO: 99) Homo sapiens cDNA, mRNA sequence 0.344344566 C22ORF33 NM_178552.2 Homo sapiens chromosome 22 open (SEQ ID NO: 100) reading frame 33 (C22orf33), mRNA. 0.344271659 HS.491292 AK093878 Homo sapiens cDNA FLJ36559 fis, (SEQ ID NO: 101) clone TRACH2009291 0.343675138 HHATL NM_020707.2 Homo sapiens hedgehog (SEQ ID NO: 102) acyltransferase-like (HHATL), mRNA. 0.342947982 GPR101 NM_054021.1 Homo sapiens G protein-coupled (SEQ ID NO: 103) receptor 101 (GPR101), mRNA. 0.342895802 HS.117299 BX110173 BX110173 NCI_CGAP_GCB1 Homo (SEQ ID NO: 104) sapiens cDNA clone IMAGp998L023103, mRNA sequence 0.341430987 HS.377021 AL832228 Homo sapiens mRNA; cDNA (SEQ ID NO: 105) DKFZp686P2136 (from clone DKFZp686P2136) 0.339934182 HIVEP2 NM_006734.3 Homo sapiens human (SEQ ID NO: 106) immunodeficiency virus type I enhancer binding protein 2 (HIVEP2), mRNA. 0.338809944 CCDC69 NM_015621.2 Homo sapiens coiled-coil domain (SEQ ID NO: 107) containing 69 (CCDC69), mRNA. 0.334035597 STK16 NM_003691.2 Homo sapiens serine/threonine kinase (SEQ ID NO: 108) 16 (STK16), transcript variant 1, mRNA. 0.333993469 HS.538083 AI191966 qe07b12.x1 Soares_testis_NHT Homo (SEQ ID NO: 109) sapiens cDNA clone IMAGE: 1738271 3, mRNA sequence 0.33374597 FLVCR2 NM_017791.1 Homo sapiens feline leukemia virus (SEQ ID NO: 110) subgroup C cellular receptor family, member 2 (FLVCR2), mRNA. 0.332804727 H2AFJ NM_177925.2 Homo sapiens H2A histone family, (SEQ ID NO: 111) member J (H2AFJ), transcript variant 1, mRNA. 0.332656128 PLEKHN1 NM_032129.1 Homo sapiens pleckstrin homology (SEQ ID NO: 112) domain containing, family N member 1 (PLEKHN1), mRNA. 0.332640503 HS.541892 AW337309 xw83e08.x1 NCI_CGAP_Pan1 Homo (SEQ ID NO: 113) sapiens cDNA clone IMAGE: 2834630 3, mRNA sequence 0.330593234 HS.580943 AW661763 hi80e01.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 114) Homo sapiens cDNA clone IMAGE: 2978616 3, mRNA sequence 0.328876547 HS.580515 R35106 yg59f10.r1 Soares infant brain 1NIB (SEQ ID NO: 115) Homo sapiens cDNA clone IMAGE: 36955 5, mRNA sequence 0.328580838 RNF122 NM_024787.2 Homo sapiens ring finger protein 122 (SEQ ID NO: 116) (RNF122), mRNA. 0.325996187 LOC100130934 XM_001716314.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 117) zinc finger protein 663 (LOC100130934), mRNA. 0.324899249 HS.128234 BX098198 BX098198 Soares_NFL_T_GBC_S1 (SEQ ID NO: 118) Homo sapiens cDNA clone IMAGp998L014015, mRNA sequence 0.321175844 HS.541852 BX115082 BX115082 Soares_NFL_T_GBC_S1 (SEQ ID NO: 119) Homo sapiens cDNA clone IMAGp998J194538, mRNA sequence 0.320655111 HS.374832 DA086964 DA086964 BRACE2 Homo sapiens (SEQ ID NO: 120) cDNA clone BRACE2041741 5, mRNA sequence 0.31731102 HS.178244 AA448469 zw80a10.s1 Soares_testis_NHT Homo (SEQ ID NO: 121) sapiens cDNA clone IMAGE: 782490 3, mRNA sequence 0.316510734 PDE7A NM_002604.1 Homo sapiens phosphodiesterase 7A (SEQ ID NO: 122) (PDE7A), transcript variant 2, mRNA. 0.315370885 HS.212582 BX642767 DKFZp781H2216_r1 781 (synonym: (SEQ ID NO: 123) hlcc4) Homo sapiens cDNA clone DKFZp781H2216 5, mRNA sequence 0.315356998 HS.584731 AA431552 zw78d11.r1 Soares_testis_NHT Homo (SEQ ID NO: 124) sapiens cDNA clone IMAGE: 782325 5 similar to contains element TAR1 repetitive element;, mRNA sequence 0.314600565 HS.584259 DR731427 MGC4.2.1.4.1.F12.F.1 NIH_MGC_331 (SEQ ID NO: 125) Homo sapiens cDNA clone MGC4.2.1.4.1.F12, mRNA sequence 0.312073933 HS.562205 BE042871 ho30a11.x1 NCI_CGAP_Lu24 Homo (SEQ ID NO: 126) sapiens cDNA clone IMAGE: 3038876 3, mRNA sequence 0.311448435 HS.580154 BU753725 UI-1-BB1p-aki-a-08-0-UI.s1 (SEQ ID NO: 127) NCI_CGAP_Pl6 Homo sapiens cDNA clone UI-1-BB1p-aki-a-08-0-UI 3, mRNA sequence 0.311068769 LOC642726 XM_931518.1 PREDICTED: Homo sapiens (SEQ ID NO: 128) hypothetical protein LOC642725, transcript variant 1 (LOC642726), mRNA. 0.311047839 HS.582893 DB338252 DB338252 TESTI2 Homo sapiens (SEQ ID NO: 129) cDNA clone TESTI2035277 3, mRNA sequence 0.311027043 DSCC1 NM_024094.1 Homo sapiens defective in sister (SEQ ID NO: 130) chromatid cohesion 1 homolog (S. cerevisiae) (DSCC1), mRNA. 0.309461784 FLJ42258 NM_001004327.1 Homo sapiens FLJ42258 protein (SEQ ID NO: 131) (FLJ42258), mRNA. 0.306433774 HS.327917 BX111675 BX111675 NCI_CGAP_Kid11 Homo (SEQ ID NO: 132) sapiens cDNA clone IMAGp998C235337, mRNA sequence 0.304704054 HS.574278 DA608330 DA608330 IMR322 Homo sapiens (SEQ ID NO: 133) cDNA clone IMR322005706 5, mRNA sequence 0.302132161 IRAK3 NM_007199.1 Homo sapiens interleukin-1 receptor- (SEQ ID NO: 134) associated kinase 3 (IRAK3), mRNA. 0.301644841 HS.128892 BX099310 BX099310 Soares_NFL_T_GBC_S1 (SEQ ID NO: 135) Homo sapiens cDNA clone IMAGp998P164537, mRNA sequence 0.301562862 HS.572999 BX111671 BX111671 NCI_CGAP_Pr28 Homo (SEQ ID NO: 136) sapiens cDNA clone IMAGp998C205228, mRNA sequence 0.301556094 LOC100129410 XM_001715712.1 PREDICTED: Homo sapiens (SEQ ID NO: 137) hypothetical protein LOC100129410 (LOC100129410), mRNA. 0.301456066 HS.407666 XM_373771 PREDICTED: Homo sapiens (SEQ ID NO: 138) hypothetical LOC388456 (LOC388456), mRNA 0.300764264 HS.544721 AI188195 qd66f02.x1 Soares_testis_NHT Homo (SEQ ID NO: 139) sapiens cDNA clone IMAGE: 1734459 3, mRNA sequence 0.29875399 HS.333785 BG196443 RST15665 Athersys RAGE Library (SEQ ID NO: 140) Homo sapiens cDNA, mRNA sequence 0.298401615 HS.527174 CF887416 UI-CF-FN0-afv-p-23-18-UI.r18 UI-CF- (SEQ ID NO: 141) FN0 Homo sapiens cDNA clone UI-CF- FN0-afv-p-23-18-UI 5, mRNA sequence 0.297928336 HS.562263 BM704202 UI-E-CK1-afj-m-16-0-UI.r1 UI-E-CK1 (SEQ ID NO: 142) Homo sapiens cDNA clone UI-E-CK1- afj-m-16-0-UI 5, mRNA sequence 0.296478585 HS.542905 AA400272 zu63c05.s1 Soares_testis_NHT Homo (SEQ ID NO: 143) sapiens cDNA clone IMAGE: 742664 3, mRNA sequence 0.296180415 LOC643894 XM_001721898.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 144) hCG1744064 (LOC643894), mRNA. 0.295414166 HS.543987 AI433683 ti88c09.x1 NCI_CGAP_Kid11 Homo (SEQ ID NO: 145) sapiens cDNA clone IMAGE: 2139088 3, mRNA sequence 0.294799162 EGOT NR_004428.1 Homo sapiens eosinophil granule (SEQ ID NO: 146) ontogeny transcript (non-protein coding) (EGOT), non-coding RNA. 0.294555566 HS.569953 BI047056 MR3-FN0206-070201-014-b05 FN0206 (SEQ ID NO: 147) Homo sapiens cDNA, mRNA sequence 0.292269527 HS.539683 AW270402 xp75b08.x1 NCI_CGAP_Ov40 Homo (SEQ ID NO: 148) sapiens cDNA clone IMAGE: 2746167 3, mRNA sequence 0.291783226 LOC100131510 XR_038622.1 PREDICTED: Homo sapiens (SEQ ID NO: 149) misc_RNA (LOC100131510), miscRNA. 0.291230816 RAB13 NM_002870.2 Homo sapiens RAB13, member RAS (SEQ ID NO: 150) oncogene family (RAB13), mRNA. 0.288870205 CPN2 XM_942072.2 PREDICTED: Homo sapiens (SEQ ID NO: 151) carboxypeptidase N, polypeptide 2, 83 kD (CPN2), mRNA. 0.288688866 SEPHS2 NM_012248.2 Homo sapiens selenophosphate (SEQ ID NO: 152) synthetase 2 (SEPHS2), mRNA. 0.288555167 HS.114286 AK025016 Homo sapiens cDNA: FLJ21363 fis, (SEQ ID NO: 153) clone COL02986 0.288413753 RAD54L2 NM_015106.2 Homo sapiens RAD54-like 2 (S. cerevisiae) (SEQ ID NO: 154) (RAD54L2), mRNA. 0.287614431 HS.560319 BC032017 Homo sapiens cDNA clone (SEQ ID NO: 155) IMAGE: 4825254 0.287002847 SC65 NM_006455.2 Homo sapiens synaptonemal complex (SEQ ID NO: 156) protein SC65 (SC65), mRNA. 0.286361546 HS.571435 AW188305 xj95a05.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 157) Homo sapiens cDNA clone IMAGE: 2664944 3, mRNA sequence 0.286179027 HS.553290 CX787363 HESC3_84_D06.g1_A036 Human (SEQ ID NO: 158) embryonic stem cells Homo sapiens cDNA clone IMAGE: 7483454 5, mRNA sequence 0.285480835 HS.545074 AA503583 ng07e12.s1 NCI_CGAP_Li1 Homo (SEQ ID NO: 159) sapiens cDNA clone IMAGE: 928750, mRNA sequence 0.283982665 LOC648370 XM_937423.1 PREDICTED: Homo sapiens (SEQ ID NO: 160) hypothetical protein LOC648370 (LOC648370), mRNA. 0.282655174 HS.571223 BX119501 BX119501 (SEQ ID NO: 161) Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone IMAGp998A084374; IMAGE: 1721743, mRNA sequence 0.280489734 HS.211821 BX090817 BX090817 NCI_CGAP_Brn52 Homo (SEQ ID NO: 162) sapiens cDNA clone IMAGp998G155675; IMAGE: 2290982, mRNA sequence 0.280225376 HS.547858 BG188175 RST7186 Athersys RAGE Library (SEQ ID NO: 163) Homo sapiens cDNA, mRNA sequence 0.278453065 PSMC3IP NM_013290.5 Homo sapiens PSMC3 interacting (SEQ ID NO: 164) protein (PSMC3IP), transcript variant 1, mRNA. 0.277366844 HS.438979 XM_374169 PREDICTED: Homo sapiens (SEQ ID NO: 165) hypothetical LOC389393 (LOC389393), mRNA 0.276716928 HUS1B NM_148959.3 Homo sapiens HUS1 checkpoint (SEQ ID NO: 166) homolog b (S. pombe) (HUS1B), mRNA. 0.276384381 LOC100129906 XM_001714161.1 PREDICTED: Homo sapiens (SEQ ID NO: 167) hypothetical protein LOC100129906 (LOC100129906), mRNA. 0.275958037 LOC100129699 XM_001718711.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 168) hCG2023449 (LOC100129699), mRNA. 0.275043965 LOC100132894 XM_001721411.1 PREDICTED: Homo sapiens (SEQ ID NO: 169) hypothetical protein LOC100132894, transcript variant 1 (LOC100132894), mRNA. 0.274523733 FBXO42 NM_018994.1 Homo sapiens F-box protein 42 (SEQ ID NO: 170) (FBXO42), mRNA. 0.274083105 PRR3 NM_001077497.1 Homo sapiens proline rich 3 (PRR3), (SEQ ID NO: 171) transcript variant 2, mRNA. 0.273505908 NPR1 NM_000906.2 Homo sapiens natriuretic peptide (SEQ ID NO: 172) receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) (NPR1), mRNA. 0.272398708 HS.90221 AK095804 Homo sapiens cDNA FLJ38485 fis, (SEQ ID NO: 173) clone FEBRA2023285 0.272299598 HS.523235 AA639731 nq82a06.s1 NCI_CGAP_Co9 Homo (SEQ ID NO: 174) sapiens cDNA clone IMAGE: 1158802 3, mRNA sequence 0.271246232 CYTH3 NM_004227.3 Homo sapiens cytohesin 3 (CYTH3), (SEQ ID NO: 175) mRNA. 0.271094707 LOC646990 XM_929962.1 PREDICTED: Homo sapiens (SEQ ID NO: 176) hypothetical protein LOC646990 (LOC646990), mRNA. 0.270098342 PDCD11 NM_014976.1 Homo sapiens programmed cell death (SEQ ID NO: 177) 11 (PDCD11), mRNA. 0.270039678 HS.543063 CD245475 AGENCOURT_14128120 (SEQ ID NO: 178) NIH_MGC_181 Homo sapiens cDNA clone IMAGE: 30374198 5, mRNA sequence 0.267719251 HS.553278 BX108660 BX108660 Soares_NFL_T_GBC_S1 (SEQ ID NO: 179) Homo sapiens cDNA clone IMAGp998K174517, mRNA sequence 0.267678157 BACH1 NM_001011545.1 Homo sapiens BTB and CNC homology (SEQ ID NO: 180) 1, basic leucine zipper transcription factor 1 (BACH1), transcript variant 3, mRNA. 0.267591499 TESSP5 NM_199183.1 Homo sapiens testis serine protease 5 (SEQ ID NO: 181) (TESSP5), mRNA. 0.265819463 HS.566643 AW135243 UI-H-BI1-ach-b-04-0-UI.s1 (SEQ ID NO: 182) NCI_CGAP_Sub3 Homo sapiens cDNA clone IMAGE: 2714071 3, mRNA sequence 0.264864487 HS.547293 BE326664 hr63a10.x1 NCI_CGAP_Kid11 Homo (SEQ ID NO: 183) sapiens cDNA clone IMAGE: 3133146 3 similar to contains MER22.t1 MER22 repetitive element;, mRNA sequence 0.264640482 LOC283849 NM_178516.2 Homo sapiens hypothetical protein (SEQ ID NO: 184) LOC283849 (LOC283849), mRNA. 0.2643875 HS.336643 CB052724 NISC_gl09g03.y1 NCI_CGAP_Lei2 (SEQ ID NO: 185) Homo sapiens cDNA clone IMAGE: 3290668 5, mRNA sequence 0.264290553 MORG1 NM_032332.2 Homo sapiens mitogen-activated protein (SEQ ID NO: 186) kinase organizer 1 (MORG1), mRNA. 0.263875482 STX4 NM_004604.3 Homo sapiens syntaxin 4 (STX4), (SEQ ID NO: 187) mRNA. 0.263609738 HS.505398 AI971548 wq87c02.x1 NCI_CGAP_GC6 Homo (SEQ ID NO: 188) sapiens cDNA clone IMAGE: 2479010 3, mRNA sequence 0.262729627 PDCD10 NM_007217.3 Homo sapiens programmed cell death (SEQ ID NO: 189) 10 (PDCD10), transcript variant 1, mRNA. 0.262668794 HS.450769 BM850770 K-EST0131410 S21SNU520 Homo (SEQ ID NO: 190) sapiens cDNA clone S21SNU520-49- E06 5, mRNA sequence 0.262479539 HS.143937 CK001807 AGENCOURT_16379396 (SEQ ID NO: 191) NIH_MGC_227 Homo sapiens cDNA clone IMAGE: 30717931 5, mRNA sequence 0.262271327 LOC653878 XM_936223.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 192) Cytosolic acyl coenzyme A thioester hydrolase, inducible (Long chain acyl- CoA thioester hydrolase) (Long chain acyl-CoA hydrolase) (CTE-I) (CTE-Ib) (LOC653878), mRNA. 0.261424332 LOC100130071 XM_001716832.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 193) GSQS6193 (LOC100130071), mRNA. 0.261375961 HS.539841 AA744242 ny62f03.s1 NCI_CGAP_GCB1 Homo (SEQ ID NO: 194) sapiens cDNA clone IMAGE: 1282877 3, mRNA sequence 0.261186788 SIPA1L1 NM_015556.1 Homo sapiens signal-induced (SEQ ID NO: 195) proliferation-associated 1 like 1 (SIPA1L1), mRNA. 0.260256467 HS.374420 CR613022 full-length cDNA clone (SEQ ID NO: 196) CS0CAP006YG05 of Thymus of Homo sapiens (human) 0.260027372 HS.545626 CD245737 AGENCOURT_14098047 (SEQ ID NO: 197) NIH_MGC_181 Homo sapiens cDNA clone IMAGE: 30376866 5, mRNA sequence 0.259158846 SPEF2 NM_024867.3 Homo sapiens sperm flagellar 2 (SEQ ID NO: 198) (SPEF2), transcript variant 1, mRNA. 0.258711041 HS.541757 CD518786 AGENCOURT_14375962 (SEQ ID NO: 199) NIH_MGC_181 Homo sapiens cDNA clone IMAGE: 30408978 5, mRNA sequence 0.258540094 C1RL NM_016546.1 Homo sapiens complement component (SEQ ID NO: 200) 1, r subcomponent-like (C1RL), mRNA. 0.258216828 LOC729157 XR_015862.1 PREDICTED: Homo sapiens (SEQ ID NO: 201) mist_RNA (LOC729157), miscRNA. 0.257210595 HS.436627 AK123174 Homo sapiens cDNA FLJ41179 fis, (SEQ ID NO: 202) clone BRACE2043036 0.2564511 HS.377419 AA532810 nf71b03.s1 NCI_CGAP_Co3 Homo (SEQ ID NO: 203) sapiens cDNA clone IMAGE: 925325 3, mRNA sequence 0.255597686 UTRN NM_007124.2 Homo sapiens utrophin (UTRN), (SEQ ID NO: 204) mRNA. 0.255569381 HS.580802 DA083801 DA083801 BRACE2 Homo sapiens (SEQ ID NO: 205) cDNA clone BRACE2037735 5, mRNA sequence 0.254989883 LOC650698 XM_939784.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 206) SH3/ankyrin domain gene 2 isoform a (LOC650698), mRNA. 0.254785157 HS.560987 BU753119 UI-1-BB1-aii-h-04-0-UI.s1 (SEQ ID NO: 207) NCI_CGAP_P15 Homo sapiens cDNA clone UI-1-BB1-aii-h-04-0-UI 3, mRNA sequence 0.254459099 HS.564967 AA725682 ai19g05.s1 Soares_testis_NHT Homo (SEQ ID NO: 208) sapiens cDNA clone 1343288 3, mRNA sequence 0.254027524 SYN2 NM_003178.4 Homo sapiens synapsin II (SYN2), (SEQ ID NO: 209) transcript variant IIb, mRNA. 0.253773675 HS.553310 CA425772 UI-H-FE1-beg-g-15-0-UI.s1 (SEQ ID NO: 210) NCI_CGAP_FE1 Homo sapiens cDNA clone UI-H-FE1-beg-g-15-0-UI 3, mRNA sequence 0.253749524 LOC651296 XM_940424.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 211) RAB, member of RAS oncogene family-like 2B isoform 1 (LOC651296), mRNA. 0.252066449 HS.543875 AL049310 Homo sapiens mRNA; cDNA (SEQ ID NO: 212) DKFZp564B206 (from clone DKFZp564B206) 0.251815933 HS.580058 AI075708 oz24f08.x1 (SEQ ID NO: 213) Soares_total_fetus_Nb2HF8_9w Homo sapiens cDNA clone IMAGE: 1676295 3, mRNA sequence 0.250190992 LOC100128695 XM_001715889.1 PREDICTED: Homo sapiens (SEQ ID NO: 214) hypothetical LOC100128695 (LOC100128695), mRNA. 0.250021556 HS.148448 AI798899 we94a12.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 215) Homo sapiens cDNA clone IMAGE: 2348734 3, mRNA sequence 0.249834606 HS.528284 AW855473 CM0-CT0275-221199-105-c11 CT0275 (SEQ ID NO: 216) Homo sapiens cDNA, mRNA sequence 0.248944004 HS.542632 U03241 Human clone pLSB8 chromosome 21 (SEQ ID NO: 217) STS 0.24758826 HS.528210 CK299576 UI-E-EJ1-ajv-n-10-0-UI.s1 UI-E-EJ1 (SEQ ID NO: 218) Homo sapiens cDNA clone UI-E-EJ1- ajv-n-10-0-UI 3, mRNA sequence 0.247471154 LOC100129147 XM_001725523.1 PREDICTED: Homo sapiens (SEQ ID NO: 219) hypothetical protein LOC100129147 (LOC100129147), mRNA. 0.24678226 C10ORF28 NM_014472.3 Homo sapiens chromosome 10 open (SEQ ID NO: 220) reading frame 28 (C10orf28), mRNA. 0.246324365 HS.559981 BQ023418 UI-1-BB1p-avd-b-12-0-UI.s1 (SEQ ID NO: 221) NCI_CGAP_P16 Homo sapiens cDNA clone UI-1-BB1p-avd-b-12-0-UI 3, mRNA sequence 0.246203021 PRAMEF7 NM_001012277.1 Homo sapiens PRAME family member (SEQ ID NO: 222) 7 (PRAMEF7), mRNA. 0.246120402 HS.537754 AW084405 xc56f12.x1 NCI_CGAP_Eso2 Homo (SEQ ID NO: 223) sapiens cDNA clone IMAGE: 2588303 3, mRNA sequence 0.245977154 HS.537638 AI610323 tp40c11.x1 NCI_CGAP_Ut4 Homo (SEQ ID NO: 224) sapiens cDNA clone IMAGE: 2190260 3, mRNA sequence 0.245420915 ANO9 NM_001012302.2 Homo sapiens anoctamin 9 (ANO9), (SEQ ID NO: 225) mRNA. 0.243992548 HS.344350 BI603074 603251294F1 NIH_MGC_96 Homo (SEQ ID NO: 226) sapiens cDNA clone IMAGE: 5303182 5, mRNA sequence 0.243782267 LOC647306 XM_930375.1 PREDICTED: Homo sapiens (SEQ ID NO: 227) hypothetical protein LOC647306 (LOC647306), mRNA. 0.24346891 CLEC18C NM_173619.2 Homo sapiens C-type lectin domain (SEQ ID NO: 228) family 18, member C (CLEC18C), mRNA. 0.242802997 HS.567436 AK026725 Homo sapiens cDNA: FLJ23072 fis, (SEQ ID NO: 229) clone LNG05713 0.24234321 HS.445121 BM545878 AGENCOURT_6505119 (SEQ ID NO: 230) NIH_MGC_125 Homo sapiens cDNA clone IMAGE: 5588241 5, mRNA sequence 0.242269316 MSTO2P NR_024117.1 Homo sapiens misato homolog 2 (SEQ ID NO: 231) pseudogene (MSTO2P), non-coding RNA. 0.242200556 LOC440459 XR_019054.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 232) solute carrier family 16, member 6 (LOC440459), miscRNA. 0.241893622 ATF6 NM_007348.2 Homo sapiens activating transcription (SEQ ID NO: 233) factor 6 (ATF6), mRNA. 0.241621226 LOC644641 XM_929614.1 PREDICTED: Homo sapiens (SEQ ID NO: 234) hypothetical protein LOC644641 (LOC644641), mRNA. 0.24116816 LOC100128392 XM_001723572.1 PREDICTED: Homo sapiens (SEQ ID NO: 235) hypothetical LOC100128392 (LOC100128392), mRNA. 0.240685997 LOC100133264 XM_001715579.1 PREDICTED: Homo sapiens (SEQ ID NO: 236) hypothetical protein LOC100133264 (LOC100133264), mRNA. 0.24037929 TP53I3 NM_004881.2 Homo sapiens tumor protein p53 (SEQ ID NO: 237) inducible protein 3 (TP53I3), transcript variant 1, mRNA. 0.239850463 LOC100132474 XM_001726593.1 PREDICTED: Homo sapiens (SEQ ID NO: 238) hypothetical protein LOC100132474 (LOC100132474), mRNA. 0.239755403 HDAC9 NM_058176.2 Homo sapiens histone deacetylase 9 (SEQ ID NO: 239) (HDAC9), transcript variant 1, mRNA. 0.239483218 HS.245812 AW130132 xf28f11.x1 NCI_CGAP_Ut1 Homo (SEQ ID NO: 240) sapiens cDNA clone IMAGE: 2619405 3, mRNA sequence 0.238382177 SLC26A8 NM_138718.1 Homo sapiens solute carrier family 26, (SEQ ID NO: 241) member 8 (SLC26A8), transcript variant 2, mRNA. 0.236865978 LOC100132485 XM_001720394.1 PREDICTED: Homo sapiens (SEQ ID NO: 242) hypothetical protein LOC100132485 (LOC100132485), mRNA. 0.236227448 RBM11 NM_144770.2 Homo sapiens RNA binding motif (SEQ ID NO: 243) protein 11 (RBM11), mRNA. 0.235733066 SLC38A5 NM_033518.1 Homo sapiens solute carrier family 38, (SEQ ID NO: 244) member 5 (SLC38A5), mRNA. 0.235692267 HS.571403 DA453004 DA453004 CTONG2 Homo sapiens (SEQ ID NO: 245) cDNA clone CTONG2025812 5, mRNA sequence 0.235571445 HS.575615 BG437415 602490627F1 NIH_MGC_18 Homo (SEQ ID NO: 246) sapiens cDNA clone IMAGE: 4622599 5, mRNA sequence 0.235449061 CBY3 XM_928982.3 PREDICTED: Homo sapiens chibby (SEQ ID NO: 247) homolog 3 (Drosophila) (CBY3), mRNA. 0.235031428 KLF3 NM_016531.4 Homo sapiens Kruppel-like factor 3 (SEQ ID NO: 248) (basic) (KLF3), mRNA. 0.234461498 EDG1 NM_001400.3 Homo sapiens endothelial (SEQ ID NO: 249) differentiation, sphingolipid G-protein- coupled receptor, 1 (EDG1), mRNA. 0.233892907 HS.576822 DB337826 DB337826 TESTI2 Homo sapiens (SEQ ID NO: 250) cDNA clone TESTI2027763 3, mRNA sequence 0.233364677 TSLP NM_138551.2 Homo sapiens thymic stromal (SEQ ID NO: 251) lymphopoietin (TSLP), transcript variant 2, mRNA. 0.233211492 HS.544326 AW510984 hd38d03.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 252) Homo sapiens cDNA clone IMAGE: 2911781 3, mRNA sequence 0.232924491 ZCCHC16 XM_937313.1 PREDICTED: Homo sapiens zinc (SEQ ID NO: 253) finger, CCHC domain containing 16 (ZCCHC16), mRNA. 0.232839494 TFEC NM_001018058.1 Homo sapiens transcription factor EC (SEQ ID NO: 254) (TFEC), transcript variant 2, mRNA. 0.232801773 HS.584251 DB298233 DB298233 BRACE2 Homo sapiens (SEQ ID NO: 255) cDNA clone BRACE2044341 3, mRNA sequence 0.232543585 HS.543175 BQ448432 UI-H-EU1-bag-d-07-0-UI.s1 (SEQ ID NO: 256) NCI_CGAP_Ct1 Homo sapiens cDNA clone UI-H-EU1-bag-d-07-0-UI 3, mRNA sequence 0.231261328 HS.538746 AW340271 hd03d12.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 257) Homo sapiens cDNA clone IMAGE: 2908439 3, mRNA sequence 0.230871518 HS.147420 AI650595 wa92h11.x1 NCI_CGAP_GC6 Homo (SEQ ID NO: 258) sapiens cDNA clone IMAGE: 2303685 3, mRNA sequence 0.230401752 HS.541581 BF589745 nac23e12.x1 Lupski_sciatic_nerve (SEQ ID NO: 259) Homo sapiens cDNA clone IMAGE: 3394270 3, mRNA sequence 0.229630597 LOC90342 XM_937145.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 260) fer-1 like protein 3 (LOC90342), mRNA. 0.229423975 HS.570176 AK094576 Homo sapiens cDNA FLJ37257 fis, (SEQ ID NO: 261) clone BRAMY2010171 0.229243683 HS.541237 AF335593 Homo sapiens cone rod homeobox (SEQ ID NO: 262) protein (CRX) mRNA, 5UTR, alternatively spliced 0.229216581 QSER1 NM_001076786.1 Homo sapiens glutamine and serine rich (SEQ ID NO: 263) 1 (QSER1), mRNA. 0.228990838 LOC646278 XR_042488.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 264) programmed cell death 6 interacting protein (LOC646278), miscRNA. 0.227506 PKLR NM_181871.2 Homo sapiens pyruvate kinase, liver and (SEQ ID NO: 265) RBC (PKLR), nuclear gene encoding mitochondrial protein, transcript variant 2, mRNA. 0.227138335 HS.578117 BQ011293 UI-1-BC1p-ary-b-02-0-UI.s1 (SEQ ID NO: 266) NCI_CGAP_P13 Homo sapiens cDNA clone UI-1-BC1p-ary-b-02-0-UI 3, mRNA sequence 0.226996944 LOC641798 XM_935965.1 PREDICTED: Homo sapiens (SEQ ID NO: 267) hypothetical protein LOC641797, transcript variant 1 (LOC641798), mRNA. 0.22641664 LOC652993 XR_018000.1 PREDICTED: Homo sapiens (SEQ ID NO: 268) hypothetical LOC652993 (LOC652993), miscRNA. 0.226005539 HS.547587 AV650212 AV650212 GLC Homo sapiens cDNA (SEQ ID NO: 269) clone GLCCCD12 3, mRNA sequence 0.224516705 HS.127874 AI809077 wf68b04.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 270) Homo sapiens cDNA clone IMAGE: 2360719 3, mRNA sequence 0.224313066 HS.537451 BU167332 AGENCOURT_7971794 (SEQ ID NO: 271) NIH_MGC_67 Homo sapiens cDNA clone IMAGE: 6169756 5, mRNA sequence 0.223606485 HS.262789 AW300868 xk07d09.x1 NCI_CGAP_Co20 Homo (SEQ ID NO: 272) sapiens cDNA clone IMAGE: 2666033 3, mRNA sequence 0.223004172 LOC100133200 XR_038343.1 PREDICTED: Homo sapiens (SEQ ID NO: 273) misc_RNA (LOC100133200), miscRNA. 0.222575995 HS.97549 BX093254 BX093254 Soares_testis_NHT Homo (SEQ ID NO: 274) sapiens cDNA clone IMAGp998H181789; IMAGE: 729281, mRNA sequence 0.222292592 LOC391429 XM_372954.1 PREDICTED: Homo sapiens (SEQ ID NO: 275) hypothetical LOC391429 (LOC391429), mRNA. 0.222241502 ATL1 NM_015915.4 Homo sapiens atlastin GTPase 1 (SEQ ID NO: 276) (ATL1), transcript variant 1, mRNA. 0.222222163 TMEM151 XM_001133285.1 PREDICTED: Homo sapiens (SEQ ID NO: 277) transmembrane protein 151 (TMEM151), mRNA. 0.221724233 HS.537742 AA609391 zu71h01.s1 Soares_testis_NHT Homo (SEQ ID NO: 278) sapiens cDNA clone IMAGE: 743473 3, mRNA sequence 0.219779366 HS.320051 AK093987 Homo sapiens cDNA FLJ36668 fis, (SEQ ID NO: 279) clone UTERU2003926 0.219380271 LOC100128300 XM_001725044.1 PREDICTED: Homo sapiens (SEQ ID NO: 280) hypothetical protein LOC100128300 (LOC100128300), mRNA. 0.219225941 TNXA NR_001284.1 Homo sapiens tenascin XA pseudogene (SEQ ID NO: 281) (TNXA) on chromosome 6. 0.21844383 HS.282153 BE501770 hw34h12.x1 NCI_CGAP_Kid11 Homo (SEQ ID NO: 282) sapiens cDNA clone IMAGE: 3184871 3, mRNA sequence 0.218362538 HS.572883 BF433017 7n23g07.x1 NCI_CGAP_Lu24 Homo (SEQ ID NO: 283) sapiens cDNA clone IMAGE: 3565621 3, mRNA sequence 0.218321837 KLHL33 XM_001714708.1 PREDICTED: Homo sapiens kelch-like (SEQ ID NO: 284) 33 (Drosophila) (KLHL33), mRNA. 0.217734316 FLJ45202 NM_207507.1 Homo sapiens FLJ45202 protein (SEQ ID NO: 285) (FLJ45202), mRNA. 0.216472558 HS.513842 BM702416 UI-E-CQ1-aez-e-05-0-UI.r1 UI-E-CQ1 (SEQ ID NO: 286) Homo sapiens cDNA clone UI-E-CQ1- aez-e-05-0-UI 5, mRNA sequence 0.216448205 LOC100133758 XM_001719929.1 PREDICTED: Homo sapiens (SEQ ID NO: 287) hypothetical LOC100133758 (LOC100133758), partial mRNA. 0.216376703 HS.436654 BM673750 UI-E-EJ0-ahh-i-07-0-UI.s1 UI-E-EJ0 (SEQ ID NO: 288) Homo sapiens cDNA clone UI-E-EJ0- ahh-i-07-0-UI 3, mRNA sequence 0.215770924 LOC730254 XM_001134396.1 PREDICTED: Homo sapiens (SEQ ID NO: 289) hypothetical LOC730254 (LOC730254), mRNA. 0.215582349 HS.371789 AI695926 ts87f05.x1 NCI_CGAP_GC6 Homo (SEQ ID NO: 290) sapiens cDNA clone IMAGE: 2238273 3, mRNA sequence 0.215499493 USP43 NM_153210.2 Homo sapiens ubiquitin-specific (SEQ ID NO: 291) peptidase 43 (USP43), mRNA. 0.215243577 ACER2 NM_001010887.2 Homo sapiens alkaline ceramidase 2 (SEQ ID NO: 292) (ACER2), mRNA. 0.21259369 HS.547601 AF086087 Homo sapiens full length insert cDNA (SEQ ID NO: 293) clone YZ84G08 0.211853505 XAGE1E NM_001097604.1 Homo sapiens X antigen family, (SEQ ID NO: 294) member 1E (XAGE1E), transcript variant 2, mRNA. 0.211840181 LOC439950 XR_040718.1 PREDICTED: Homo sapiens (SEQ ID NO: 295) misc_RNA (LOC439950), miscRNA. 0.211785217 HS.514440 AW440458 xt14e05.x1 NCI_CGAP_Ut4 Homo (SEQ ID NO: 296) sapiens cDNA clone IMAGE: 2779136 3, mRNA sequence 0.211422576 HS.539450 AA810581 oa85h04.s1 NCI_CGAP_GCB1 Homo (SEQ ID NO: 297) sapiens cDNA clone IMAGE: 1319095 3, mRNA sequence 0.211265945 HS.566937 BF509831 UI-H-BI4-apc-c-04-0-UI.s1 (SEQ ID NO: 298) NCI_CGAP_Sub8 Homo sapiens cDNA clone IMAGE: 3086958 3, mRNA sequence 0.210924998 ZNF155 NM_003445.2 Homo sapiens zinc finger protein 155 (SEQ ID NO: 299) (ZNF155), transcript variant 1, mRNA. 0.210528175 HS.566801 BF514696 UI-H-BW1-anh-g-12-0-UI.s1 (SEQ ID NO: 300) NCI_CGAP_Sub7 Homo sapiens cDNA clone IMAGE: 3082558 3, mRNA sequence 0.209096858 HS.539384 AI733668 ov06f07.x5 NCI_CGAP_Kid3 Homo (SEQ ID NO: 301) sapiens cDNA clone IMAGE: 1636549 3, mRNA sequence 0.209048271 LOC100128675 NR_024562.1 Homo sapiens hypothetical (SEQ ID NO: 302) LOC100128675 (LOC100128675), transcript variant 2, non-coding RNA. 0.207726738 HS.543828 BE646122 7e81f05.x1 NCI_CGAP_Pr28 Homo (SEQ ID NO: 303) sapiens cDNA clone IMAGE: 3288897 3, mRNA sequence 0.207339851 LOC151300 NR_015390.1 Homo sapiens hypothetical LOC151300 (SEQ ID NO: 304) (LOC151300), transcript variant 2, non- coding RNA. 0.20716108 LOC441179 XR_017855.1 PREDICTED: Homo sapiens (SEQ ID NO: 305) hypothetical gene supported by AK055887; AK125190 (LOC441179), misc RNA. 0.206908794 KCNN3 NM_002249.4 Homo sapiens potassium (SEQ ID NO: 306) intermediate/small conductance calcium-activated channel, subfamily N, member 3 (KCNN3), transcript variant 1, mRNA. 0.206836678 CCR9 NM_031200.1 Homo sapiens chemokine (C-C motif) (SEQ ID NO: 307) receptor 9 (CCR9), transcript variant A, mRNA. 0.20675256 HS.581662 BF514549 UI-H-BW1-ang-b-09-0-UI.s1 (SEQ ID NO: 308) NCI_CGAP_Sub7 Homo sapiens cDNA clone IMAGE: 3081953 3, mRNA sequence 0.206187298 HS.438365 BX105017 BX105017 (SEQ ID NO: 309) Soares_fetal_heart_NbHH19W Homo sapiens cDNA clone IMAGp998A20842, mRNA sequence 0.205884997 PROP1 NM_006261.2 Homo sapiens PROP paired-like (SEQ ID NO: 310) homeobox 1 (PROP1), mRNA. 0.205797283 GM127 XM_938555.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 311) RIKEN 2610020C11 (gm127), mRNA. 0.204046436 LOC100128918 XM_001718466.1 PREDICTED: Homo sapiens (SEQ ID NO: 312) hypothetical protein LOC100128918 (LOC100128918), mRNA. 0.20382379 PTK9 NM_198974.1 Homo sapiens PTK9 protein tyrosine (SEQ ID NO: 313) kinase 9 (PTK9), transcript variant 2, mRNA. 0.203738347 LOC644763 XM_927860.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 314) erythrocyte membrane protein band 4.1 like 4B isoform 2 (LOC644763), mRNA. 0.203340973 OR2W3 NM_001001957.2 Homo sapiens olfactory receptor, family (SEQ ID NO: 315) 2, subfamily W, member 3 (OR2W3), mRNA. 0.202444303 HS.534997 L23402 Human (clone Z149) retinal mRNA (SEQ ID NO: 316) 0.202329789 HS.575831 BF849882 PM4-EN0063-151100-002-b09 EN0063 (SEQ ID NO: 317) Homo sapiens cDNA, mRNA sequence 0.200264582 HS.580053 DA724733 DA724733 NT2RI3 Homo sapiens (SEQ ID NO: 318) cDNA clone NT2RI3006220 5, mRNA sequence 0.200160695 HS.177532 BX355045 BX355045 Homo sapiens (SEQ ID NO: 319) NEUROBLASTOMA COT 25- NORMALIZED Homo sapiens cDNA clone CS0DC029YI23 5-PRIME, mRNA sequence 0.19853518 LOC649596 XM_938659.1 PREDICTED: Homo sapiens similar to (SEQ ID NO: 320) zinc finger protein 329 (LOC649596), mRNA. 0.194110351 LOH12CR1 NM_058169.2 Homo sapiens loss of heterozygosity, (SEQ ID NO: 321) 12, chromosomal region 1 (LOH12CR1), mRNA. 0.190833594 LOC642420 XM_930795.1 PREDICTED: Homo sapiens (SEQ ID NO: 322) hypothetical protein LOC642420 (LOC642420), mRNA. 0.190190067 HS.560563 BF510793 UI-H-BI4-apo-a-02-0-UI.s1 (SEQ ID NO: 323) NCI_CGAP_Sub8 Homo sapiens cDNA clone IMAGE: 3088010 3, mRNA sequence 0.18973138 LOC388743 XM_938885.2 PREDICTED: Homo sapiens similar to (SEQ ID NO: 324) calpain 8, transcript variant 4 (LOC388743), mRNA. 0.185853814 HS.561542 AW873324 hl92a07.x1 NCI_CGAP_Lu24 Homo (SEQ ID NO: 325) sapiens cDNA clone IMAGE: 3009396 3, mRNA sequence 0.185133299 HS.278145 AW629128 hi51d04.x1 Soares_NFL_T_GBC_S1 (SEQ ID NO: 326) Homo sapiens cDNA clone IMAGE: 2975815 3, mRNA sequence 0.181685962 CTF1 NM_001330.2 Homo sapiens cardiotrophin 1 (CTF1), (SEQ ID NO: 327) mRNA. 0.178257498 HS.563654 BG201663 RST21004 Athersys RAGE Library (SEQ ID NO: 328) Homo sapiens cDNA, mRNA sequence 0.174248793 SLC25A26 NM_001009938.1 Homo sapiens solute carrier family 25, (SEQ ID NO: 329) member 26 (SLC25A26), transcript variant 3, mRNA. 0.172335203 CNTNAP5 NM_130773.2 Homo sapiens contactin associated (SEQ ID NO: 330) protein-like 5 (CNTNAP5), mRNA. 0.151445464 LOC100131972 XM_001715773.1 PREDICTED: Homo sapiens (SEQ ID NO: 331) hypothetical protein LOC100131972 (LOC100131972), mRNA. −0.260428493 FAM10A4 NR_002183.1 Homo sapiens family with sequence (SEQ ID NO: 332) similarity 10, member A4 pseudogene (FAM10A4), non-coding RNA. −0.265794765 DCTD NM_001921.2 Homo sapiens dCMP deaminase (SEQ ID NO: 333) (DCTD), transcript variant 2, mRNA. −0.267394612 HNRNPR NM_005826.3 Homo sapiens heterogeneous nuclear (SEQ ID NO: 334) ribonucleoprotein R (HNRNPR), transcript variant 2, mRNA. −0.268053209 SNORD31 NR_002560.1 Homo sapiens small nucleolar RNA, (SEQ ID NO: 335) C/D box 31 (SNORD31), small nucleolar RNA. −0.270868488 LOC100130919 XM_001722872.1 PREDICTED: Homo sapiens (SEQ ID NO: 336) hypothetical protein LOC100130919 (LOC100130919), mRNA. −0.271399854 GLOD4 NM_016080.2 Homo sapiens glyoxalase domain (SEQ ID NO: 337) containing 4 (GLOD4), mRNA. −0.272886738 LOC100128266 XR_037888.1 PREDICTED: Homo sapiens (SEQ ID NO: 338) misc_RNA (LOC100128266), miscRNA. −0.284403431 LOC100129697 XM_001732822.1 PREDICTED: Homo sapiens (SEQ ID NO: 339) hypothetical protein LOC100129697 (LOC100129697), mRNA. −0.295431965 TMX3 NM_019022.3 Homo sapiens thioredoxin-related (SEQ ID NO: 340) transmembrane protein 3 (TMX3), mRNA. −0.29953729 YBX1 NM_004559.3 Homo sapiens Y box binding protein 1 (SEQ ID NO: 341) (YBX1), mRNA. −0.308386487 IL18BP NM_173042.2 Homo sapiens interleukin 18 binding (SEQ ID NO: 342) protein (IL18BP), transcript variant A, mRNA. −0.334319636 HSP90AB1 NM_007355.2 Homo sapiens heat shock protein 90 kDa (SEQ ID NO: 343) alpha (cytosolic), class B member 1 (HSP90AB1), mRNA. −0.382917922 LOC439953 XR_017375.2 PREDICTED: Homo sapiens (SEQ ID NO: 344) misc_RNA (LOC439953), miscRNA. −0.406810782 COL9A2 NM_001852.3 Homo sapiens collagen, type IX, alpha 2 (SEQ ID NO: 345) (COL9A2), mRNA. −0.416050903 FKBP1A NM_054014.1 Homo sapiens FK506 binding protein (SEQ ID NO: 346) 1A, 12 kDa (FKBP1A), transcript variant 12A, mRNA. −0.451735283 HSPA1A NM_005345.4 Homo sapiens heat shock 70 kDa (SEQ ID NO: 347) protein 1A (HSPA1A), mRNA. −0.452374174 LOC286157 XR_038597.1 PREDICTED: Homo sapiens (SEQ ID NO: 348) misc_RNA (LOC286157), miscRNA. −0.454880106 HNRPA1L-2 NR_002944.2 Homo sapiens heterogeneous nuclear (SEQ ID NO: 349) ribonucleoprotein A1 pseudogene (HNRPA1L-2), non-coding RNA. −0.506093336 LOC643873 XR_039149.1 PREDICTED: Homo sapiens (SEQ ID NO: 350) misc_RNA (LOC643873), miscRNA. −0.525288782 NBPF14 NM_015383.1 Homo sapiens neuroblastoma (SEQ ID NO: 351) breakpoint family, member 14 (NBPF14), mRNA. −0.525590589 LOC643287 XM_928075.2 PREDICTED: Homo sapiens similar to (SEQ ID NO: 352) prothymosin alpha, transcript variant 1 (LOC643287), mRNA. −0.590984232 LOC728755 XM_001128377.2 PREDICTED: Homo sapiens similar to (SEQ ID NO: 353) hCG1984907 (LOC728755), mRNA. −0.597068853 ALDH1A1 NM_000689.3 Homo sapiens aldehyde dehydrogenase (SEQ ID NO: 354) 1 family, member A1 (ALDH1A1), mRNA. −0.639014352 ALDH1A1 NM_000689.3 Homo sapiens aldehyde dehydrogenase (SEQ ID NO: 354) 1 family, member A1 (ALDH1A1), mRNA. −0.804986707 HLA-DRB6 NR_001298.1 Homo sapiens major histocompatibility (SEQ ID NO: 355) complex, class II, DR beta 6 (pseudogene) (HLA-DRB6), non-coding RNA. −0.933938319 FCER1A NM_002001.2 Homo sapiens Fc fragment of IgE, high (SEQ ID NO: 356) affinity I, receptor for; alpha polypeptide (FCER1A), mRNA.

The predictive power of the signature is shown by its excellent sensitivity and specificity. As can be seen in a ROC analysis (FIG. 1), both these parameters are remarkably high.

It is clear that these data support our working hypothesis that PBM are indeed “imprinted” by the tumor, and that there is a subset of genes showing altered expression in monocytes in patients with cancer compared to healthy controls.

Example 2 Independent Validation of the Diagnostic Signature

To further optimize and validate the signature, a qRT-PCR analysis was undertaken to confirm the expression profile data. Instead of the full list of genes, the list was pruned to the combined genes of the two limma signatures. This was done because these signatures showed high overlap and each separately performed similar to the merged signature. Thus, it was reasoned that this more limited set of genes would perform similarly. qPCR was chosen because expression changes can be more easily picked up in this analysis. The list of genes selected for this analysis is shown in Table II.

TABLE II Genes selected for further qRT-PCR analysis, with indication of regulation in cancer settings based on the microarray data. Up- or down- regulated in cancer as compared Gene to healthy abbreviation Definition control ADM Homo sapiens adrenomedullin (ADM), Up mRNA. CTSZ Homo sapiens cathepsin Z (CTSZ), Up mRNA. HBA1 Homo sapiens hemoglobin, alpha 1 Up (HBA1), mRNA. HBB Homo sapiens hemoglobin, beta (HBB), Up mRNA. TAF15 Homo sapiens TAF15 RNA polymerase Up II, TATA box binding protein (TBP)- associated factor, 68kDa (TAF15), mRNA. ACP5 acid phosphatase 5, tartrate resistant Up ALDH1A1 Homo sapiens aldehyde dehydrogenase Up for primary 1 family, member Al (ALDH1A1), tumors; down mRNA. for metastasized tumors APP amyloid beta (A4) precursor protein Up ENSA Endosulfine alpha Up for primary tumors; no significant difference with healthy control for metastasized tumors CCR1 Homo sapiens chemokine (C-C motif) Up receptor 1 (CCR1), mRNA. CD68 macrophage antigen CD68 (GP110, Up LAMP4, SCARD1, macrosialin) DDIT4 Homo sapiens DNA-damage-inducible Up transcript 4 (DDIT4), mRNA. LOC100170833 PREDICTED: Homo sapiens Up hypothetical protein LOC100170833, mRNA. FCER1A Homo sapiens Fc fragment of IgE, high Down affinity I, receptor for; alpha polypeptide (FCER1A), mRNA. HLA-DRB4 Homo sapiens major histocompatibility Up complex, class II, DR beta 4 (pseudogene). IER2 Homo sapiens immediate early response Up 2 (IER2), mRNA. LAPTM4A lysosomal protein transmembrane 4 Up alpha (MBNT, HUMORF13) SEPT5 Homo sapiens septin 5 (SEPT5), Up mRNA. TKT Homo sapiens transketolase (Wernicke- Up Korsakoff syndrome) (TKT), mRNA. ARPC1B actin related protein 2/3 complex, Up subunit 1B, 41 kDa BAX Homo sapiens BCL2-associated X Up protein (BAX), mRNA. DNAJC7 Homo sapiens DnaJ (Hsp40) homolog, Up subfamily C, member 7 (DNAJC7), mRNA. FKBP5 Homo sapiens FK506 binding protein 5 Up (FKBP5), mRNA. GPER Homo sapiens G protein-coupled Up estrogen receptor 1 (GPER), mRNA. HMOX1 Homo sapiens heme oxygenase Up (decycling) 1 (HMOX1), mRNA. HP Homo sapiens haptoglobin (HP), Up mRNA. IL1R2 interleukin 1 receptor, type II Up S100P Homo sapiens S100 calcium binding Up protein P (S100P), mRNA. SLC39A1 Homo sapiens solute carrier family 39 Up (zinc transporter), member 1 (SLC39A1), mRNA. SOCS3 Homo sapiens suppressor of cytokine Up signaling 3 (SOCS3), mRNA. HNRNPK Homo sapiens heterogeneous nuclear Up ribonucleoprotein K (HNRNPK), mRNA. RILPL2 Rab interacting lysosomal protein-like 2 Up TNPO1 Homo sapiens transportin 1 (TNPO1), Up mRNA. HLA-DQA1 major histocompatibility complex, class Up II, DQ alpha 1 LOC100170939 Homo sapiens glucuronidase, beta Up pseudogene (LOC100170939), non- coding RNA. LOC644063 PREDICTED: Homo sapiens Up hypothetical protein LOC644063 (LOC644063), mRNA. LOC723972 Homo sapiens hepatopoietin PCn127 Up (LOC723972), non-coding RNA. RN28S1 RNA, 28S ribosomal 1 Up SDHC succinate dehydrogenase complex, Up subunit C, integral membrane protein, 15 kDa CCL18 chemokine (C-C motif) ligand 18 Up (pulmonary and activation-regulated) TNF tumor necrosis factor Up for primary tumors; down for metastasized tumors CXCL9 chemokine (C-X-C motif) ligand 9 Down SLPI secretory leukocyte peptidase inhibitor Up CXCR4 chemokine (C-X-C motif) receptor 4 Up

For this analysis, a second aliquot of the material used for the Illumina analysis was taken for random eight persons out of every group (HV, P, PM). From the stored lysed monocytes in buffer RLT, the RNA was freshly extracted.

mRNA expression levels for selected genes in Table II are shown in FIG. 2. It can be seen that the qRT-PCR analysis confirms the findings by Illumina, and these genes show indeed altered expression in the groups with non-metastatic or metastatic cancer as compared to the healthy control group. As can be expected, the effect is not as pronounced for every gene. In this regard, it has to be taken into account that the sample size for the RT-PCR (n=8) is considerably smaller than that for the Illumina analysis. Also, there is a slight (quantitative) difference when samples are normalized for beta-2-microglobulin levels or for actin levels, although this does not affect the (qualitative) direction of the effect. For four markers, the altered expression in disease setting differs between the Illumina and qPCR analysis. This can be due to a number of factors: sensitivities of the methodology, specificity of the Illumina probes in comparison to the qPCR assays, the fact that Illumina analysis combines P and PM groups, a different normalization used, etc. The qPCR data are believed to be correct (since qPCR is a technique better suited to detect changes in expression), and these markers are in principle reliable. However, according to particular embodiments, these four genes (i.e., DNAJC7, LOC723972, RN28S1, and TAF15) are not used as a monocyte expression marker.

It is striking that a number of genes show such significantly altered expression that their expression alone retains high predictive power, as can, e.g., be seen from the area under curve graph. This is for instance true for DDIT4 (AUC: 0.9609), ADM (AUC: 0.9453), GPER (AUC: 0.9297), S100P (AUC: 0.9141) and SOCS3 (AUC: 0.9141). So expression of one of these genes alone can even be used as marker. Of note, although these are the best performing ones as sole markers, they are not exceptional. Also FKBP5, IL1R2, FCER1A and HBA1 for instance show AUC values of over 0.85; and it is likely that further analysis of markers may reveal that more of the 336 genes can be used as single markers.

Interestingly, the qRT-PCR analysis also shows that some particular markers also make the distinction between the P and PM patient groups, possibly due to over-amplification of expression differences that were non-significant in the microarray data. This is, e.g., the case for the ACP5, ADM, ALDH1A1, APP, CD68, ENSA, FKBP5, GPER, HLA-DQA1, LOC644063, LOC723972, RN28S1, S100P, TAF15, TKT and TNF markers. Most particularly envisaged are markers selected from the list of: ACP5, ALDH1A1, ENSA, LOC644063, LOC723972, TAF15, TKT and TNF; as these markers have a very low p value when comparing expression values in monocytes from patients with non-metastasized versus metastasized tumors.

In case of ACP5, ADM, FKBP5, GPER, HLA-DQA1, LOC723972, RN28S1, S100P, and TAF 15 the difference between the HV and P groups is further increased between the HV and PM groups—the expression level thus is a direct indication of whether the patient is in the P or PM group. Note that the increase in difference applies both for markers that are up-regulated as for markers whose expression is down-regulated (e.g., HLA-DQA1, LOC723972, TAF15)—in this case, an increased difference means a further down-regulation.

In case of other markers like CD68, TKT and LOC644063 the difference (e.g., increase) between P and HV is larger than that between the PM and HV groups (e.g., in case of increased levels in P vs. HV, levels in PM are decreased compared to P—see also Table III). There may not always be a significant difference anymore between the PM and HV groups, but there remains a significant difference between the levels in the combined P, PM groups and the HV group. In some cases, like APP and ENSA, there is a significant difference between HV and P groups, but the trend reversal is so strong that there is no significant difference any more between HV and PM or the combined P, PM groups (using a significance cut-off value of p=0.1). Whereas these markers alone would not be prime candidates to discriminate between PM and HV, they can be used in combination with other markers that can establish the presence of cancer (i.e., indicate that the patient is either P or PM). The levels of these markers can then discriminate between the P and PM groups. In other words, when the presence of cancer is established, these markers can be used to classify the cancer as metastatic or non-metastatic. They are also useful as sole marker to detect non-metastasized cancer (but not as sole marker to detect metastasized cancer).

In case of ALDH1A1 and TNF, the difference between the HV and P groups is quite small, but there is a very significant difference between the P and PM group. This marker can thus be used together with other markers that can discriminate between P, PM and HV but not necessarily between P and PM groups to check whether a patient has a primary or metastasized tumor.

An overview of the markers shown in FIG. 2 is incorporated in Table III, including parameters that show their value as disease and progression marker.

TABLE III Some relevant parameters for the genes shown in FIG. 2. Up, down, same: indicates whether expression levels for the gene product marker are up-regulated, down-regulated or not significantly changed, respectively, between the groups listed in the column header. p HV-total cancer (P, AUC for PM ROC P vs. PM vs. PM gene groups) p P-PM curve HV HV vs. P ACP5 0.0081 0.0465 0.7891 Up up up ADM 0.0941 0.3974 0.9453 up up up ALDH1A1 0.9624 0.0376 0.5078 up down down APP 0.1576 0.0812 0.7344 up up down BAX 0.0978 0.289 0.6719 up up down CD68 0.064 0.1913 0.7578 up up down CTSZ 0.0637 0.2237 0.7578 up up down CXCR4 0.0065 0.794 0.8417 up up same DDIT4 0.001 0.8452 0.9609 up up same DNAJC7 0.5431 0.7796 0.6172 down down same ENSA 0.258 0.0148 0.6719 up same down FCER1A 0.0768 0.2687 0.7266 down down down FKBP5 0.0013 0.1028 0.8906 up up up GPER 0.0138 0.0887 0.9297 up up up HBA1 0.0861 0.8154 0.8594 up up same HBB 0.0773 0.9122 0.8482 up up same HLA- 0.0918 0.1113 0.7656 down down down DQA1 HMOX1 0.0729 0.6284 0.6953 up up same HP 0.0218 0.554 0.7578 up up same IL1R2 0.0288 0.4311 0.8672 up up same LAPTM4A 0.0206 0.701 0.7813 up up same LOC644063 0.0415 0.1058 0.7143 up up down LOC723972 0.9645 0.1874 0.5625 down down down RN28S1 0.5908 0.3764 0.5781 down down down S100P 0.0003 0.1891 0.9141 up up up SOCS3 0.0081 0.413 0.9141 up up up TAF15 0.3084 0.0344 0.6484 down down down TKT 0.0061 0.0297 0.8203 up up down TNF 0.7325 0.0257 0.5313 same/up down down

All markers of Table III are also shown in FIG. 2.

Markers with a p value below 0.1 (or even more particularly below 0.05) in the first column are very reliable disease markers, as this is an indication how different values from the combined P and PM groups are from the values obtained from the HV control group.

Markers with a p value below 0.1 (or even more particularly below 0.05) in the second column are particularly suited as markers to discriminate between non-metastasized and metastasized tumors.

Markers with an AUC higher than 0.8 (or even more particularly higher than 0.85) are markers that are particularly suited as single markers: these markers combine a high sensitivity with a high specificity.

The three columns to the right indicate whether the marker is up- or down-regulated, or not significantly changed (“same”) when comparing the two mentioned groups. This can be used to correlate the expression levels with presence of disease, or with the presence of metastasis (disease stage).

Example 3 Validation of the Expression of the PBM-Associated Predictive Biomarkers During Early Stages of Tumor Development and Tumor Progression in Mouse Models of CRC

To further characterize the biological role of the candidate genes, it was investigated if the human PBM gene profile can be extended to animal models of CRC. Chemically induced carcinogenesis models in Balb/c mice were used for disease follow-up from the initiation period. The azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model of colon cancers results in a predictable tumorigenesis that closely resembles the progression of the human disease (Tanaka et al., 2003). Five-week-old male Balb/c mice were injected intraperitoneally with a single dose (10 mg/kg/body weight) of the carcinogen AOM on day 1 followed by three cycles of administration of the inflammation-inducing DSS into their drinking water (3% solution) on day 1 to 5, day 22 to 26 and day 43 to 47. After this treatment, the colon of the treated mice reveals aberrant crypt foci (ACF), dysplastic lesions, adenomas and adenocarcinomas. Peripheral blood was collected and PBM were isolated by FACS using the specific mouse monocyte marker CD115 for the generation of a mouse monocyte library for CRC and gene expression studies. At the end-stage, peripheral blood was collected and the colon was subjected to histopathological evaluation (Tanaka et al., 2003). A different cohort of mice was only injected with DSS to induce chronic colitis without malignant transformation to confirm the disease specificity of the set of candidate genes in mice. As shown in FIG. 3, gene expression in mice indeed follows the same pattern as that observed in humans.

Example 4 Multicentric Validation of the Biomarker Panel

The genetic signature defined in example 1 was further confirmed in a broad test set of patient samples acquired in a multicentric trial involving a number among the major oncological centers throughout the EU: Leuven (Belgium), Brussels (Belgium), Heidelberg (Germany) and Rome (Italy). In each center, PBMs were isolated from both patients and healthy volunteers using the protocol as described in Example 1. Blood processing (isolation of monocytes) was performed within 2 hours of isolation of the peripheral blood. Lysed monocytes were stored in RLT buffer while awaiting analysis. Validation of a number of samples from the different centers (n=76 for patients and 65 for healthy subjects; matched for age, gender and ethnicity) was done i.a. using the qRT-PCR assay described in example 2, and is still ongoing for further samples. 43 genes were selected for confirmation analysis: the 29 genes listed in Table III and 14 additional markers. The list of genes is depicted in Table IV. Complete statistical analysis is still ongoing, but at least for a first set of genes evaluated in this setting, the results could be confirmed (these genes include ACP5, ADM, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, FKBP5, GPER, HBA1, HBB, HP, LAPTM4A, S100P, SEPT5 and TKT). Representative examples are shown in FIGS. 4A and 4B. Interestingly, and although there is inherently more variability in this data set due to the different centers and persons involved in sample gathering and monocyte isolation (which explains the slightly lower AUC-values in the ROC curves in FIG. 4), the data reach statistical significance when all results are combined, but also when the data are assessed center by center (e.g., comparing only patients from Leuven with healthy volunteers from Leuven). This indicates the robustness of the method, i.e., it is independent from the center were the isolation took place or the person who isolated the monocytes.

For statistical analyses between the patients (P), metastatic patients (PM) and healthy subjects (HV) groups, data sets were randomly split in a training and test set (⅔ for training, ⅓ for test set).

Two classification approaches were used: SGMV (single gene majority vote, to evaluate the contribution of single genes to the signature) and SVM (support vector machine). Also, three different quantification approaches were evaluated: absolute (i.e., copies of gene of interest per 10,000 copies of B2M), relative (each individual expression value normalized to the average of the HV group) and dCT: (delta-CT of gene of interest—reference gene).

Both classifications and the three quantification approaches yield very similar results. Representative ROC values for both classification approaches are shown in FIG. 5. Interestingly, a subset of 12 genes selected from the 43 genes of Table IV (here: ADM, BAX, CD68, CTSZ, CXCR4, FKBP5, GPER, HBA1, HBB, LAPTM4A, S100P and TKT) performed equally well: there is virtually no difference in the ROC curve, AUC or BER values (compare left and right panel of FIG. 5). This indicates that, once a certain number of genes is reached to make up the signature, no sensitivity or specificity is added anymore. Also, it confirms the strength of these genes as single markers (see, e.g., also the ROC curves in FIG. 4B) or when combining a low number of them.

The expression values of the 12 genes for the subset are shown in FIG. 6.

TABLE IV List of 43 genes evaluated in the multicentric validation experiment. ACP5 ADM APP BAX CD68 CTSZ CXCR4 DDIT4 FCER1A FKBP5 GPER HBA1 HBB HP IL1R2 LAPTM4A LOC644063 S100P SLPI SOCS3 TKT TNF ALDH1A1 ARPC1B CCR1 DNAJC7 ENSA HLA-DQ1 HLA-DRB4 HMOX1 HNRNPK LOC100008589 LOC10017093 LOC643888 LOC723972 RLPL2 RN28S1 SDHC SEPT5 SLC39A1 TAF15 TNPO1 IER2

Example 5 Assessment of a Tumor-Driven Monocyte Education Toward “Malignancy”

The entirely new and exciting concept of education of monocytes by the tumor in an endocrine manner was investigated in vitro and in vivo.

5.1 Specificity of the Signature for Colorectal Cancer Vs. Other Cancers

To evaluate whether the altered gene expression of the signature genes in monocytes is specific to colorectal cancer or a general hallmark of cancer, monocytes were also isolated from gastric cancer patients, and expression of selected genes was evaluated (FIG. 7). Remarkably, most genes that show a clear difference in expression in colorectal cancer (vs. healthy volunteers) are expressed at a similar level in gastric cancer patients and healthy volunteers (e.g., ACP5, CXCR4). Some of the genes also show an altered expression pattern, but not to the same extent as in colorectal cancer (e.g., DDIT4, FKBP5, S100P). This is a clear indication that the marker genes are specific markers for colorectal cancer and the differential expression is not due to the general presence of a tumor. Without being bound to a particular mechanism, it does make sense that colorectal cancers will secrete different factors than gastric cancers, thereby eliciting a differential response in monocytes.

To explore this further, PBM from healthy volunteers were isolated and incubated with medium conditioned by either colorectal tumor cell line HCT116 or the gastric tumor cell line MKN (FIG. 8). As can be seen from this figure, incubation with colorectal tumor-conditioned medium induced the monocytes to alter their expression pattern to that observed in monocytes isolated from colorectal cancer patients (see also Example 5.2). Incubation with gastric tumor-conditioned medium had a smaller effect on gene expression for the genes analyzed, and the observed expression pattern is significantly different from that observed for monocytes incubated with colorectal tumor-conditioned medium (FIG. 8).

5.2 Specificity of the Signature for Cancer Versus Non-Cancerous Colon Cells.

In order to assess whether the altered expression pattern in monocytes is specific to the presence of a tumor or could be due to factors from the colonic epithelium, PBM from healthy volunteers were isolated and incubated for 18 hours with medium conditioned by either colorectal tumor cell line HCT116 or the benign colonic epithelial cell line CCD 841 CoN and evaluated for the expression of the set of candidate genes. A representative selection of these genes is shown in FIG. 9. Monocytes with mock medium or medium conditioned by colonic epithelial cells have a very similar expression pattern, while monocytes incubated with tumor-conditioned medium show a significant different expression pattern for these genes. Interestingly, this effect is independent from hypoxia, as there is no significant difference in expression pattern between monocytes incubated with tumor-conditioned medium in normal or hypoxic conditions. This further illustrates the specificity of the colorectal cancer gene signature of these monocytes. Moreover, this may provide an explanation why the present methods are particularly suited to detect tumors at an early stage (before they become hypoxic). Indeed, the altered gene expression is dependent on the nature of the tumor (even a small tumor), and not on the presence of hypoxia.

5.3 the Gene Signature can be Reversed In Vitro

We have also evaluated if the genetic signature of CRC PBM can be reversed. For this purpose, PBM from healthy volunteers were incubated with CCD or HCT-conditioned medium for 18 hours (as described in example 5.2), after which the medium was refreshed to plain culture medium, and the gene signature was assessed after 72 hours. See FIG. 10. Interestingly, the altered expression of the genes from monocytes exposed to HCT116-conditioned medium completely reverted to baseline values (i.e., those found in monocytes that have never been exposed to cancer) when no longer exposed to tumor-conditioned medium, indicating that the presence of tumor-derived factors is necessary to maintain the differential expression. This plasticity of the gene signature has important practical consequences: it makes the marker panel ideally suited to monitor both the success of therapy, and the potential recurrence of a tumor. Indeed, after, e.g., surgical resection to remove the tumor, expression of the genes described herein can be checked to see whether the tumor has indeed been completely removed, as the gene expression should return to baseline values. For follow-up, this expression can be monitored, as altered expression of these genes will indicate recurrence of the colorectal cancer. Of course, this expression can be monitored both in patients in remission wherein expression of the signature was measured during disease stage, as well as in patients wherein expression of the signature has not been evaluated before.

Example 6 Further Validation in Mouse Models

As a further follow-up, the tumor promoting potential of these educated monocytes will be investigated in vivo in a short-term four-week-long model of aberrant crypt foci formation, in which tumorigenesis is induced by weekly injection of the carcinogen AOM. By coinjecting PBM from healthy volunteers or CRC patients, respectively, in the highly immunodeficient mouse strain NOD.Cg-Prkdcscid II2rgtm1Wjl/SzJ (The Jackson Laboratory) we will assess the tumor-promoting potential of these monocytes by analyzing the number and severity of aberrant crypt foci. Immunohistological analysis of tumor tissue and the infiltrating monocytes at different stages will reveal the interaction of the monocytes with the tumor.

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1. A method of diagnosing colorectal cancer in a subject, the method comprising: determining the expression of at least one gene product in a sample of monocytes from the subject.
 2. The method of claim 1, wherein the at least one gene product is not a gene product that is used as a marker to identify the nature of monocytes in said sample.
 3. The method according to claim 1, wherein the monocytes were circulating monocytes.
 4. The method according to claim 1, wherein altered expression of the at least one gene product is indicative of cancer in the subject.
 5. The method according claim 1, wherein at least one gene product comprises at least one product is selected from the group of genes listed in Table I.
 6. The method according to claim 1, wherein at least one gene product comprises at least one product selected is from a gene listed in Table II, Table III and/or Table IV.
 7. The method according to claim 1, wherein at least one gene product comprises at least one product from a gene selected from the group consisting of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SEPT5, SOCS3, TAF15, TKT, and TNF.
 8. The method according to claim 1, wherein the at least one gene product is at least two gene products.
 9. The method according to claim 8, wherein at least one gene product is from a gene selected from the group consisting of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SEPT5, SOCS3, TAF15, TKT, and TNF; and wherein at least one other gene product is selected from the group of genes listed in Tables I, II, III and/or IV.
 10. A method of discriminating primary from metastatic cancer in a subject, the method comprising: determining the expression of two or more gene products in a sample of monocytes from the subject, wherein at least one of the gene products is from a gene selected from the group consisting of ACP5, ADM, ALDH1A1, APP, BAX, CD68, ENSA, FKBP5, GPER, HLA-DQA1, LOC644063, LOC723972, RN28S1, S100P, TAF15, TKT and TNF; and at least one other gene product is from a gene selected from the group of genes listed in Tables I, II, III and/or IV.
 11. The method according to claim 1, wherein the cancer is in Tis or T1 stage.
 12. The method according to claim 1, wherein the monocytes have been isolated from a whole blood sample.
 13. A method of diagnosing cancer in a subject, the method comprising the steps of: determining the expression or levels of expression of at least one gene product by forming a complex between said gene product and a molecule that specifically binds with said gene product and measuring the complex in a monocyte population from a blood sample isolated from the subject; and correlating the expression or levels of expression of the at least one gene product with the presence or absence of cancer in the subject.
 14. The method according to claim 13, wherein the blood sample is obtained from the subject no more than 12 hours prior to the determining step.
 15. The method according to claim 1, wherein the expression levels are determined with RT-PCR.
 16. The method according to claim 1, further comprising monitoring recurrence of a cancer in the subject.
 17. The method according to claim 1, further comprising monitoring response to therapy in the subject.
 18. The method according to claim 1, further comprising altering treatment of the subject based on the results of the determination.
 19. A gene signature comprising at least two genes, wherein at least one gene is selected from the group consisting of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SEPT5, SOCS3, TAF15, TKT, and TNF; and at least one other gene is selected from a gene listed in Tables I, II, III and/or IV.
 20. The gene signature according to claim 19, which is a monocyte gene signature.
 21. A medicament comprising: the gene signature according to claim
 19. 22. A diagnostic comprising: the gene signature according to claim
 19. 23. A kit comprising: means for detecting expression or levels of expression of at least one gene product of at least one gene selected from the group consisting of ACP5, ADM, ALDH1A1, APP, BAX, CD68, CTSZ, CXCR4, DDIT4, DNAJC7, ENSA, FCER1A, FKBP5, GPER, HBA1, HBB, HLA-DQA1, HMOX1, HP, IL1R2, LAPTM4A, LOC644063, LOC723972, RN28S1, S100P, SEPT5, SOCS3, TAF15, TKT, and TNF; and means for detecting expression or level of expression of at least one gene product of at least one gene selected from the group of genes listed in Tables I, II, III and/or IV.
 24. (canceled)
 25. The kit according to claim 23, wherein the means for detecting expression comprise primers or probes for the selected genes.
 26. The method according to claim 8, wherein the at least one gene product is at least five gene products.
 27. The method according to claim 12, wherein the monocytes are from a peripheral blood sample or a central blood sample.
 28. The method according to claim 7, further comprising: altering the subject's treatment based upon the determination.
 29. The method according to claim 13, further comprising: altering the subject's treatment based upon the diagnosis. 